Animal models and therapeutic molecules

ABSTRACT

The invention discloses methods for the generation of chimaeric human-non-human antibodies and chimaeric antibody chains, antibodies and antibody chains so produced, and derivatives thereof including fully humanised antibodies; compositions comprising said antibodies, antibody chains and derivatives, as well as cells, non-human mammals and vectors, suitable for use in said methods.

CROSS REFERENCE

This application is a continuation of U.S. Ser. No. 16/216,666, filedDec. 11, 2018, which is a continuation of U.S. Ser. No. 15/088,805,filed Apr. 1, 2016, which is a continuation of PCT/GB2014/052971, filedOct. 1, 2014, which claims the benefit of FR1359518 filed Oct. 1, 2013and of GB1317410.7 filed Oct. 1, 2013, the contents of each of which areincorporated by reference herein in their entirety.

BACKGROUND

The present invention relates inter alia to non-human animals and cellsthat are engineered to contain exogenous DNA, such as humanimmunoglobulin gene DNA, their use in medicine and the study of disease,methods for production of non-human animals and cells, and antibodiesand antibody chains produced by such animals and derivatives thereof.

SUMMARY OF THE INVENTION

All nucleotide co-ordinates for the mouse are those corresponding toNCBI m37 for the mouse C57BL/6J strain, e.g. April 2007 ENSEMBL Release55.37h, e.g. NCBI37 July 2007 (NCBI build 37) (e.g. UCSC version mm9 seeWorld Wide Web (www) genome.ucsc.edu and World Wide Web (www)genome.ucsc.edu/FAQ/FAQ/releases.html) unless otherwise specified. Humannucleotides coordinates are those corresponding to GRCh37 (e.g. UCSCversion hg 19, World Wide Web (www)genome.ucsc.edu/FAQ/FAQreleases.html), February 2009 ENSEMBL Release55.37, or are those corresponding to NCBI36, Ensemble release 54 unlessotherwise specified. Rat nucleotides are those corresponding to RGSC 3.4Dec. 2004 ENSEMBL release 55.34w, or Baylor College of Medicine HGSC v3.4 Nov. 2004 (e.g., UCSC rn4, see World Wide Web (www) genome.ucsc.eduand World Wide Web (www) genome.ucsc.edu/FAQ/FAQreleases.html) unlessotherwise specified. Reference to work in mice herein is by way ofexample only, and reference to mice is taken to include reference to allnon-human mammals unless otherwise apparent from the disclosure, withmice being preferred as the non-human mammal.

The disclosures of US2012/0204278 and PCT/GB2013/050682 are incorporatedherein by reference. All definitions disclosed in US2012/0204278 andPCT/GB2013/050682 are specifically and explicitly disclosed herein.

Reference to human gene segments herein encompasses both the germlinehuman gene segment sequence or the recombined form of the gene segmentthat can include one or more mutations relative to the germline humangene segment sequence, for example alleles disclosed in the IMGTdatabase and 1000 Genomes database, as well as in WO2013041844 (suchalleles and their sequences being explicitly incorporated herein byreference).

All gene segments referred to herein can be identified using standardsequence analysis by comparison to human germline gene segment sequencesoptionally by reference to the public sequence databases, such as theIMGT or 1000 Genomes database.

In one aspect the invention relates to a non-human vertebrate (e.g., amouse or rat) or cell whose genome comprises human VH, D and JH genesegments upstream of a constant region at a heavy chain locus and/orhuman VL and JL gene segments upstream of a constant region at a lightchain locus, wherein the gene segments are operably linked to theconstant region thereof so that the vertebrate or cell is capable ofexpressing immunoglobulin heavy and/or light chains comprising human VHand VL domains respectively, wherein the heavy chain locus comprises ahuman 01 allele VH gene segment capable of recombining with a human Dand JH gene segment to produce a VH domain, wherein the light chainlocus comprises a human 01 allele VL gene segment capable of recombiningwith a human JL gene segment to produce a VL domain, or wherein the cellcan develop into a vertebrate that expresses said heavy and/or lightchains.

As explained further in the examples below, the inventors havesurprisingly shown that human 01 alleles can be used to produceantigen-specific binding sites, wherein these are properly recombined ina non-human vertebrate, display junctional and somatic mutations and canbe properly expressed and isolated.

In another aspect the invention relates to a non-human vertebrate orcell (eg, a mouse cell or rat cell) whose genome comprises (a) humanJH2*01 and/or human JH6*01 or JH6*02 and/or JH3*02, one or more human VHgene segments and one or more human D gene segments upstream of aconstant region at a heavy chain locus and/or (b) human JK2*01 and/orhuman JK4*01 and one or more human VK gene segments upstream of aconstant region at a light chain locus, wherein the gene segments ineach locus are operably linked to the constant region thereof so thatthe vertebrate or cell is capable of producing an antibody heavy chainand an antibody light chain, or where the cell can develop into avertebrate that expresses an antibody heavy chain and an antibody lightchain, wherein the heavy chain is produced by recombination of the humanJH2*01 and/or JH6*01 or JH6*02 segment and/or JH3*02 with a D segmentand a VH segment and the light chain is produced by recombination of thehuman Jk2*01 and/or Jk4*01 segment with a VK segment. In an example, thegenome comprises human JH2*01. In an example, the genome comprises humanJH2*01 and JH6*01. In an example, the genome comprises human JH2*01,JH6*01 and JH3*02. In an example, the genome comprises human JH6*01. Inan example, the genome comprises human JH6*01 and JH3*02. In an example,the genome comprises human JH3*02. In an example, the heavy chain isproduced by recombination of the human JH2*01 segment with a D segmentand a VH segment. In an example, the heavy chain is produced byrecombination of the human JH6*01 segment with a D segment and a VHsegment. In an example, the heavy chain is produced by recombination ofthe human JH6*02 segment with a D segment and a VH segment. In anexample, the heavy chain is produced by recombination of the humanJH3*02 segment with a D segment and a VH segment.

In another aspect the invention relates to a non-human vertebrate whosegenome comprises (i) human JH1*01, JH2*01, JH3*02, JH4*02, JH5*02 and/orJH6*01 or JH6*02, one or more human VH gene segments and one or morehuman D gene segments upstream of a constant region at a heavy chainlocus and/or (ii) human Jk1*01, Jk2*01, Jk3*01, Jk4*01 and/or Jk5*01 andone or more human VK gene segments upstream of a constant region at alight chain locus, wherein the gene segments in each locus are operablylinked to the constant region thereof so that the vertebrate or cell iscapable of producing an antibody heavy chain and an antibody lightchain, or where the cell can develop into a vertebrate that expresses anantibody heavy chain and/or an antibody light chain, wherein the heavychain is produced by recombination of the human JH1*01, JH2*01, JH3*02,JH4*02, JH5*02 and/or JH6*01 or JH6*02 segment with a D segment and a VHsegment and the light chain is produced by recombination of the humanJk1*01, Jk2*01, Jk3*01, Jk4*01 and/or Jk5*01 segment with a VK segment.In an example, the genome comprises human JH1*01, JH2*01, JH3*02,JH4*02, JH5*02 and JH6*01. In an example, the genome comprises humanJH1*01, JH2*01, JH3*02, JH4*02, JH5*02 and JH6*02. In an example, theheavy chain is produced by recombination of the human JH2*01 segmentwith a D segment and a VH segment. In an example, the heavy chain isproduced by recombination of the human JH6*01 segment with a D segmentand a VH segment. In an example, the heavy chain is produced byrecombination of the human JH6*02 segment with a D segment and a VHsegment. In an example, the heavy chain is produced by recombination ofthe human JH3*02 segment with a D segment and a VH segment. Additionallyor alternatively to these heavy chain examples, in an example the lightchain is produced by recombination of the human JK2*01 segment with a VKsegment.

In one embodiment, the non-human vertebrate further comprises one ormore of the VH gene segments and/or one or more of the D gene segmentsfrom Table 7 and/or one or more VK gene segments from Table 12. In afurther embodiment, the non-human vertebrate further comprises the VHgene segments and D gene segments from Table 3 and/or the VK genesegments from Table 10 or 11.

The segments described herein have been identified by the inventors asbeing widely and highly used across diverse human ethnic populations,and thus widely tolerated for antibody generation and efficacy in humansin general.

In a further embodiment, the invention relates to a non-human vertebrateor vertebrate cell (e.g. a mouse cell or rat cell) whose genomecomprises one or more human VH gene segments, one or more human JH genesegments and one or more human D gene segments upstream of a constantregion at a heavy chain locus and one or more human JL gene segments andone or more human VL gene segments upstream of a constant region at alight chain locus, wherein the gene segments in each locus are operablylinked to the constant region thereof so that the vertebrate or cell iscapable of producing an antibody heavy chain and an antibody lightchain, or where the cell can develop into a vertebrate that expresses anantibody heavy chain and an antibody light chain, wherein said one ormore human VH gene segments of the heavy chain locus comprise or consistof one, more or all human VH gene segments selected from the groupconsisting of VH3-23*04, VH7-4-1*01, VH4-4*02, VH1-3*01, VH3-13*01,VH3-7*01, VH3-20*d01 and VH3-9*01.

In a further embodiment, the invention relates to a non-human vertebrateor vertebrate cell (e.g. a mouse cell or rat cell) whose genomecomprises one or more human VH gene segments, one or more human JH genesegments and one or more human D gene segments upstream of a constantregion at a heavy chain locus and one or more human JK gene segments andone or more human VK gene segments upstream of a constant region at alight chain locus, wherein the gene segments in each locus are operablylinked to the constant region thereof so that the veretebrate or cell iscapable of producing an antibody heavy chain and an antibody lightchain, or where the cell can develop into a vertebrate that expresses anantibody heavy chain and an antibody light chain, wherein said one ormore human VK gene segments comprise or consist of one, more or allhuman VK gene segments selected from the group consisting of Vκ4-1*01,Vκ2-28*01, Vκ1D-13*d01, Vκ1-12*01, Vκ1D-12*02, Vκ3-20*01, Vκ1-17*01,Vκ1D-39*01, Vκ3-11*01, Vκ1D-16*01 and Vκ1-9*d01.

In another embodiment the invention relates to a non-human vertebrate orvertebrate cell (eg, a mouse cell or rat cell) whose genome compriseshuman JH2*01 and/or human JH6*02, one or more human VH gene segments andone or more human D gene segments upstream of a constant region at aheavy chain locus and/or human Jk2*01 and/or human Jk4*01 and one ormore human VK gene segments upstream of a constant region at a lightchain locus, wherein the gene segments in each locus are operably linkedto the constant region thereof so that the cell or vertebrate is capableof producing an antibody heavy chain and an antibody light chain, orwhere the cell can develop into a vertebrate that expresses an antibodyheavy chain and an antibody light chain, wherein the heavy chain isproduced by recombination of the human JH2*01 and/or JH6*02 segment witha D segment and a VH segment and/or the light chain is produced byrecombination of the human Jk2*01 and/or Jk4*01 segment with a VKsegment;

wherein said one or more human VH gene segments of the heavy chain locuscomprise or consist of one, more or all human VH gene segments selectedfrom the group consisting of VH3-23*04, VH7-4-1*01, VH4-4*02, VH1-3*01,VH3-13*01, VH3-7*01 and VH3-20*d01 and/or

wherein said one or more human VK gene segments comprise or consist ofone, more or all human VH gene segments selected from the groupconsisting of Vκ4-1*01, VK2-28*01, Vκ1D-13*d01, Vκ1-12*01, Vκ1D-12*02,Vκ3-20*01, Vκ1-17*01, Vκ1D-39*01, Vκ3-11*01, Vκ1D-16*01 and Vκ1-9*d01.

Optionally reference to a human gene segment herein is the recombinedform of the gene segment.

As explained further in the examples below, the inventors havesurprisingly shown that heavy and/or light chains produced according tothe invention can be used to produce antigen-specific binding sites,wherein these are properly recombined in a non-human vertebrate, displayjunctional and somatic mutations and can be properly expressed andisolated.

In another aspect the invention relates to a non-human vertebrate (eg, amouse or rat) or cell whose genome comprises an Ig gene segmentrepertoire produced by targeted insertion of human Ig gene segments intoone or more endogenous Ig loci, the genome comprising human Vλ and Jλgene segments upstream of a constant region, wherein the human Vλ and Jλgene segments are selected from one, more or all of the segments ofTable 18 and have been provided by insertion into an endogenous lightchain locus of the vertebrate or cell, wherein the vertebrate comprisesimmunoglobulin light chains comprising lambda variable regions (lambdalight chains) or the cell can develop into a vertebrate that expressessaid immunoglobulin light chains, wherein the lambda light chainscomprise immunoglobulin light chains comprising lambda variable regionsrecombinants of one, more or all of the human Vλ and Jλ gene segments ofTable 18.

Endogenous Light Chain Inactivation & High Expression of Human LambdaVariable Regions in Transgenic Non-Human Vertebrates & Cells

As explained further in the examples below, the inventors havesurprisingly observed very high expression levels of light chainscomprising human lambda variable regions (at least 70 or 80% human Vlambda) from transgenic light chain loci produced by targeted insertionof human lambda gene segments into endogenous non-human vertebrate lightchain loci. This is possible even in the presence of endogenousnon-human vertebrate V and J gene segments in the vertebrate genome.Also, the surprisingly high levels of expression are achieved wheninsertion of human lambda gene segments are in the endogenous kappa orlambda locus. Such high levels by targeted insertion has not hithertobeen published in the art.

The inventors also surprisingly observed that endogenous kappa chainexpression can be completely inactivated by targeted insertion of humanlambda gene sequence into the endogenous kappa locus, as explainedfurther in the examples.

The targeted insertion of human gene segments into endogenous Ig loci isadvantageous because it enables the operable location of inserted humanIg sequences with respect to endogenous Ig constant regions andendogenous control regions, such as enhancers and other locus controlregions for example. Thus, targeted insertion allows one to harnessendogenous control important in one or more of Ig gene segmentrecombination, allelic exclusion, affinity maturation, class switching,levels of Ig expression and desirable development of the B-cellcompartment. As such, targeted insertion is superior to early attemptsin the art to produce transgenic Ig loci and expression, which attemptsrelied on the introduction into non-human vertebrate cells of vectorssuch as YACs bearing human Ig gene segments. YACs are randomlyintegrated into the vertebrate cell genome, so that it is difficult toachieve the control provided by targeted insertion and the concomitantbenefits that are brought in terms of harnessing endogenous controlmechanisms. In addition, random insertion often results in the insertedhuman Ig gene segments coming under the control of heterologous controlelements and/or epigenetic chromosomal modifications such as methylationand chromatin confirmations, either of which can be detrimental toproper Ig gene segment recombination, allelic exclusion, affinitymaturation, class switching, levels of Ig expression and desirabledevelopment of the B-cell compartment. Random insertion typicallyresults in 2 or more copies of the introduced transgene which can causechromosomal instability and therefore result in poor breedingperformance of the animals in addition to detrimental effects on properIg gene segment recombination, allelic exclusion, affinity maturation,class switching, levels of Ig expression and desirable development ofthe B-cell compartment. Thus, prior art attempts using random insertionhave tended to lead to poor B-cell development, relatively small B-cellcompartments and inferior Ig expression and a concomitant difficulty inisolating an antibody with a desired characteristic.

The invention therefore provides the following aspects:—

Expression of Human Lambda Variable Regions

Every embodiment of the invention disclosed herein can be put intopractice with a specific lambda allele disclosed in Table 18, or anycombination thereof. Where gene segments are referred to herein withoutreference to a specific allele, these can optionally be the specificalleles disclosed in any one of Tables 1 to 18.

-   1. A non-human vertebrate (eg, a mouse or rat) whose genome    comprises an Ig gene segment repertoire produced by targeted    insertion of human Ig gene segments into one or more endogenous Ig    loci, the genome comprising human Vλ and Jλ gene segments upstream    of a constant region, wherein the human Vλ and Jλ gene segments have    been provided by insertion into an endogenous light chain locus of    the vertebrate, wherein the vertebrate expresses immunoglobulin    light chains comprising lambda variable regions (lambda light    chains), wherein the lambda light chains comprise immunoglobulin    light chains comprising lambda variable regions derived from    recombination of human Vλ and Jλ gene segments.

A non-human vertebrate (eg, a mouse or rat) whose genome comprises an Iggene segment repertoire produced by targeted insertion of human Ig genesegments into one or more endogenous Ig loci, the genome comprisinghuman Vλ and Jλ gene segments upstream of a constant region, wherein thehuman Vλ and Jλ gene segments have been provided by insertion into anendogenous light chain locus of the vertebrate, wherein the vertebrateexpresses immunoglobulin light chains comprising lambda variable regions(lambda light chains), and wherein at least 70 or 80% of the variableregions of the lambda light chains expressed by the vertebrate arederived from recombination of human Vλ and Jλ gene segments. This isdemonstrated in the examples below.

For example, at least 70, 75, 80, 84, 85, 90, 95, 96, 97, 98 or 99%, or100% of the variable regions of the lambda light chains expressed by thevertebrate are derived from recombination of human Vλ and Jλ genesegments. This is demonstrated in the examples below.

In embodiments, there is provided

A non-human vertebrate ES cell (eg, a mouse ES cell or rat ES cell)whose genome comprises an Ig gene segment repertoire produced bytargeted insertion of human Ig gene segments into one or more endogenousIg loci, the genome comprising human Vλ and Jλ gene segments upstream ofa constant region, wherein the human Vλ and Jλ gene segments have beenprovided by insertion into an endogenous light chain locus of thevertebrate cell, wherein the cell can develop into a vertebrate thatexpresses immunoglobulin light chains comprising lambda variable regions(lambda light chains), wherein the lambda light chains compriseimmunoglobulin light chains comprising lambda variable regions derivedfrom recombination of human Vλ and Jλ gene segments.

A non-human vertebrate ES cell (eg, a mouse ES cell or rat ES cell)whose genome comprises an Ig gene segment repertoire produced bytargeted insertion of human Ig gene segments into one or more endogenousIg loci, the genome comprising human Vλ and Jλ gene segments upstream ofa constant region, wherein the human Vλ and Jλ gene segments have beenprovided by insertion into an endogenous light chain locus of thevertebrate cell, wherein the cell can develop into a vertebrate thatexpresses immunoglobulin light chains comprising lambda variable regions(lambda light chains), and wherein at least 70 or 80% (for example, atleast 70, 75, 80, 84, 85, 90, 95, 96, 97, 98 or 99%, or 100%) of thevariable regions of the lambda light chains expressed by the vertebrateare derived from recombination of human Vλ and Jλ gene segments.

In an example, surprisingly expression of immunoglobulin light chainscomprising lambda variable regions derived from recombination of humanVλ and Jλ gene segments is achieved even when the genome comprisesendogenous non-human vertebrate lambda variable region gene segments(eg, endogenous Vλ and/or Jλ gene segments, optionally a completeendogenous repertoire of Vλ and Jλ gene segments). Thus, in an example,the genome comprises endogenous non-human vertebrate lambda variableregion gene segments (eg, endogenous Vλ and/or Jλ gene segments,optionally a complete endogenous repertoire of Vλ and Jλ gene segments).In another example, such endogenous gene segments are absent from thegenome.

-   2. The vertebrate or cell of aspect 1, optionally wherein the human    Vλ and Jλ insertion comprises at least the functional human V and J    gene segments (optionally also human CA) comprised by a human lambda    chain Ig locus from Vλ2-18 to Cλ7. In one example, the insertion    also comprises lambda inter-gene segment sequences. These are human    sequences or they can be sequences of the non-human vertebrate    species (eg, where the vertebrate is a mouse, sequences between    corresponding mouse lambda gene segments can be used).

In one embodiment, the V and J gene segments are the alleles of Table18. In a further embodiment, the CA gene segments are the allelesdisclosed in Table 18.

-   3. The vertebrate or cell of aspect 1 or 2, optionally wherein the    genome is homozygous for the human Vλ and Jλ gene segment insertion    and endogenous kappa chain expression in said vertebrate is    substantially or completely inactive. In one example, less than 10,    5, 4, 3, 2, 1 or 0.5% of light chains are provided by endogenous    kappa chains (ie, kappa chains whose variable regions are derived    from recombination of non-human vertebrate V and J gene segments).-   4. The vertebrate or cell of any preceding aspect, optionally    wherein the endogenous locus is an endogenous kappa locus.-   5. The vertebrate or cell of any preceding aspect, optionally    wherein the endogenous locus is an endogenous lambda locus.    ≥60% of all light chains have human lambda V regions-   6. A non-human vertebrate (eg, a mouse or rat) whose genome    comprises an Ig gene segment repertoire produced by targeted    insertion of human Ig gene segments into one or more endogenous Ig    loci, the genome comprising (i) human Vλ and Jλ gene segments    upstream of a constant region, wherein the human Vλ and Jλ gene    segments have been provided by insertion into an endogenous light    chain locus of the vertebrate and (ii) kappa V gene segments    upstream of a constant region, wherein the vertebrate expresses    immunoglobulin light chains comprising human lambda variable regions    (human lambda light chains), and wherein at least 60% of the light    chains expressed by the vertebrate are provided by said human lambda    light chains. This is demonstrated in the examples below.

For example, at least 65, 70, 80, 84, 85, 90, 95, 96, 97, 98 or 99%, or100% of the light chains expressed by the vertebrate are provided bysaid human lambda light chains. For example, at least 84% of the lightchains expressed by the vertebrate are provided by said human lambdalight chains. For example, at least 95% of the light chains expressed bythe vertebrate are provided by said human lambda light chains. This isdemonstrated in the examples below.

In one embodiment, there is provided a non-human vertebrate ES cell (eg,a mouse ES cell or rat ES cell) whose genome comprises an Ig genesegment repertoire produced by targeted insertion of human Ig genesegments into one or more endogenous Ig loci, the genome comprising (i)human Vλ and Jλ gene segments upstream of a constant region, wherein thehuman Vλ and Jλ gene segments have been provided by insertion into anendogenous light chain locus of the vertebrate and (ii) kappa V genesegments upstream of a constant region, wherein the cell can developinto a vertebrate that expresses immunoglobulin light chains comprisinghuman lambda variable regions (human lambda light chains), and whereinat least 60% of the light chains expressed by the vertebrate areprovided by said human lambda light chains.

-   7. A non-human vertebrate or a non-human vertebrate cell (eg, a    mouse, rat, mouse cell or a rat cell) whose genome comprises an Ig    gene segment repertoire produced by targeted insertion of human Ig    gene segments into one or more endogenous Ig loci, the genome    comprising a targeted insertion of human immunoglobulin Vλ and Jλ    gene segments into an endogenous non-human vertebrate light kappa or    lambda chain locus downstream of endogenous VL and JL gene segments    for expression of light chains comprising human lambda variable    regions; wherein the human Vλ and Jλ insertion comprises at least    the functional human V and J (and optionally also functional human    CA) gene segments comprised by a human lambda chain Ig locus from    Vλ2-18 to Cλ7.

As demonstrated in the examples, endogenous light chain expression fromsaid locus is inactivated and also human lambda variable regionexpression dominates over endogenous lambda variable region expression.

By “downstream” is meant 3′ of the gene segments on the same chromosome.In one example, the endogenous V and J gene segments are inverted withrespect to the human gene segments and optionally moved out of theendogenous light chain locus. In one example, the human gene segmentsare downstream of all of the endogenous V and J segments of said kappaor lambda locus. The possibility of retaining the endogenous V-Jsequences and intergenic sequences is advantageous since embeddedcontrol regions and/or genes are retained that may be desirable in thevertebrate.

Optionally the insertion also comprises lambda inter-gene segmentsequences. These are human sequences or they can be sequences of thenon-human vertebrate species (eg, where the vertebrate is a mouse,sequences between corresponding mouse lambda gene segments can be used).

Expression of VJCλ Lambda Chains

-   8. A non-human vertebrate or a non-human vertebrate cell (eg, a    mouse, rat, mouse cell or a rat cell) whose genome comprises an Ig    gene segment repertoire produced by targeted insertion of human Ig    gene segments into one or more endogenous Ig loci, the genome    comprising a targeted insertion of human immunoglobulin VA, Jλ and    CA genes into an endogenous non-human vertebrate kappa or lambda    light chain locus upstream of an endogenous non-human vertebrate    kappa or lambda constant region for expression of a human VJC light    chain; optionally wherein the human VJC insertion comprises at least    the functional human V, J and C gene segments comprised by a human    lambda chain Ig locus from Vλ3-1 to Cλ7 (eg, comprised by a human    lambda chain Ig locus from 2-18 to Cλ7).

In one embodiment, the V and J gene segments are the alleles of Table18. In a further embodiment, the CA gene segments are the allelesdisclosed in Table 18.

As demonstrated in the examples, human lambda variable region expressiondominates over endogenous kappa variable region expression. Endogenouskappa chain expression from the endogenous locus can be inactivated.

Optionally the insertion also comprises lambda inter-gene segmentsequences. These are human sequences or they can be sequences of thenon-human vertebrate species (eg, where the vertebrate is a mouse,sequences between corresponding mouse lambda gene segments can be used).

-   9. A non-human vertebrate or a non-human vertebrate cell (eg, a    mouse, rat, mouse cell or a rat cell) whose genome comprises an Ig    gene segment repertoire produced by targeted insertion of human Ig    gene segments into one or more endogenous Ig loci, the genome    comprising a targeted insertion of at least the functional human Vλ    and Jλ (and optionally human functional Cλ) gene segments comprised    by a human lambda chain Ig locus from Vλ3-1 to Cλ7 (optionally from    Vλ2-18 to Cλ7, further optionally the specific alleles of Table 18)    into an endogenous non-human vertebrate kappa light chain locus    downstream of the mouse VK and JK gene segments for expression of a    light chain comprising a human lambda variable region, whereby in    the presence of said insertion expression of endogenous kappa light    chains derived from said mouse VK and JK gene segments is    substantially or completely inactivated.

In one example, less than 10, 5, 4, 3, 2, 1 or 0.5% of light chains areprovided by endogenous kappa chains (ie, kappa chains whose variableregions are derived from recombination of non-human vertebrate VK and JKgene segments).

Optionally the insertion also comprises lambda inter-gene segmentsequences. These are human sequences or they can be sequences of thenon-human vertebrate species (eg, where the vertebrate is a mouse,sequences between corresponding mouse lambda gene segments can be used).

-   10. A non-human vertebrate or a non-human vertebrate cell (eg, a    mouse, rat, mouse cell or a rat cell), wherein in the genome of    which the mouse IgK-VJ has been moved away from the mouse EK    enhancer, thereby inactivating endogenous IgK-VJ regions. This is    demonstrated in the examples.-   11. The vertebrate of cell of aspect 10, optionally wherein the    IgK-VJ has been moved away from the mouse Eκ enhancer by insertion    of human VL and JL gene segments between the mouse IgK-VJ and the Eκ    enhancer; optionally wherein the insertion is an insertion as    recited in any preceding aspect 1-9 or an insertion of human VK and    JK gene segments.-   12. The vertebrate or cell of any preceding aspect, optionally    wherein the human Vλ and Jλ gene segments have been inserted within    100, 75, 50, 40, 30, 20, 15, 10 or 5 kb of an endogenous non-human    vertebrate light chain enhancer. In one example, the enhancer is a    lambda enhancer (eg, mouse Eλ2-4, Eλ4-10 or Eλ3-1) when the    insertion is into an endogenous lambda locus. In one example, the    enhancer is a kappa enhancer (eg, iEκ or 3′Eκ) when the insertion is    into an endogenous kappa locus.-   13. The vertebrate or cell of any preceding aspect, optionally    wherein the human Vλ and Jλ gene segments are provided in the genome    by the targeted insertion of at least 10 human Vλ gene segments with    human Jλ gene segments upstream of an endogenous non-human    vertebrate light chain constant region of said light chain locus.    For example, the human gene segments are provided by insertion of at    least a portion of a human Ig lambda chain locus from Vλ2-18 to    Vλ3-1; or at least a portion of a human Ig lambda chain locus from    Vλ2-18 to Vλ3-1 inserted with Jλ1, Jλ2, Jλ3, Jλ6 and Jλ7; or at    least a portion of a human Ig lambda chain locus from Vλ2-18 to Cλ7    (optionally excluding Jλ4Cλ4 and/or Jλ5Cλ5).

Optionally at least 2, 3, 4 or 5 human Jλ are inserted. In oneembodiment, the inserted JAs are different from each other. For example,human Jλ1, Jλ2, Jλ3, Jλ6 and Jλ7 are inserted, optionally as part ofrespective human JλCλ clusters.

Optionally a human light chain enhancer, eg Eλ, is inserted. Forexample, insertion of human EA between the human Jλ segments and theendogenous constant region; or between human CA gene segments (whenthese are inserted) and the endogenous constant region.

-   14. The vertebrate or cell of any preceding aspect, optionally    wherein the lambda light chains provide a repertoire of human lambda    variable regions derived from human Vλ gene segments Vλ3-1 and    optionally one or more of Vλ2-18, Vλ3-16, V2-14, Vλ3-12, Vλ2-11,    Vλ3-10, Vλ3-9, Vλ2-8 and Vλ4-3 that have been provided in the genome    by targeted insertion into said light chain locus.

This is useful because Vλ3-1 is a highly-used lambda gene segment inhumans (FIG. 59; Ignatovich et al 1997) and thus it is desirable thatcells and vertebrates of the invention provide for the inclusion oflambda variable regions based on this gene segment for selection againstantigen, particularly for the development of antibody therapeutics forhuman use.

-   15. The vertebrate or cell of any preceding aspect, optionally    wherein the lambda light chains provide a repertoire of human lambda    variable regions derived from human Vλ gene segments Vλ2-14 and one    or more of Vλ2-18, Vλ3-16, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8,    Vλ4-3 and Vλ3-1 that have been provided in the genome by targeted    insertion into said light chain locus.

This is useful because Vλ2-14 is a highly-used lambda gene segment inhumans and thus it is desirable that cells and vertebrates of theinvention provide for the inclusion of lambda variable regions based onthis gene segment for selection against antigen, particulary for thedevelopment of antibody therapeutics for human use.

The vertebrate or cell of any preceding aspect, optionally wherein thelambda light chains provide a repertoire of human lambda variableregions derived from human Vλ gene segments Vλ2-8 and one or more ofVλ2-18, Vλ3-16, V2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 andVλ3-1 that have been provided in the genome by targeted insertion intosaid light chain locus.

This is useful because Vλ2-8 is a highly-used lambda gene segment inhumans and thus it is desirable that cells and vertebrates of theinvention provide for the inclusion of lambda variable regions based onthis gene segment for selection against antigen, particulary for thedevelopment of antibody therapeutics for human use.

The vertebrate or cell of any preceding aspect, optionally wherein thelambda light chains provide a repertoire of human lambda variableregions derived from human Vλ gene segments Vλ3-10 and one or more ofVλ2-18, Vλ3-16, V2-14, Vλ3-12, Vλ2-11, Vλ2-14, Vλ3-9, Vλ2-8, Vλ4-3 andVλ3-1 that have been provided in the genome by targeted insertion intosaid light chain locus.

This is useful because Vλ3-10 is a highly-used lambda gene segment inhumans and thus it is desirable that cells and vertebrates of theinvention provide for the inclusion of lambda variable regions based onthis gene segment for selection against antigen, particulary for thedevelopment of antibody therapeutics for human use.

-   16. The vertebrate or cell of any preceding aspect, optionally    wherein the human Vλ gene segments comprise the functional Vλ    comprised by a human lambda chain Ig locus from Vλ2-18 to Vλ3-1.

For example, the human Vλ gene segments comprise at least human V genesegment Vλ3-1 or at least segments Vλ2-18, Vλ3-16, V2-14, Vλ3-12,Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 and Vλ3-1.

-   17. The vertebrate of any preceding aspect, optionally wherein the    vertebrate expresses more lambda chains than kappa chains. Lambda    chains comprise variable regions derived from recombination of Vλ    and Jλ gene segments—for example, expressed with a lambda constant    region. Kappa chains comprise variable regions derived from    recombination of VK and JK gene segments—for example, expressed with    a kappa constant region.-   18. The vertebrate of any preceding aspect, optionally wherein the    vertebrate expresses no endogenous kappa chains. For example,    endogenous kappa chain expression can be inactivated by any of the    means described herein, such as by inversion of all or part of the    endogenous kappa VJ region or by insertion of a marker (eg, neo) or    other interfering sequence in an endogenous kappa locus (a locus not    comprising human lambda gene segments according to the invention).-   19. The vertebrate of any preceding aspect, optionally wherein kappa    chain expression is substantially or completely inactive in said    vertebrate. In one example, less than 10, 5, 4, 3, 2, 1 or 0.5% of    light chains are provided by kappa chains.-   20. The vertebrate or cell of any preceding aspect, optionally    wherein a human EA enhancer is inserted in said endogenous non-human    vertebrate locus. For example, there is inserted a human 5′ MAR and    human EA (and optionally the human 3′ MAR) in germline    configuration. For example, there is inserted a sequence    corresponding to the human lambda intronic region immediately 3′ of    human Jλ7-Cλ7 to, and including, at least the human EA (and    optionally also the human 3′ MAR)—optionally including at least 30    kb of intronic region 3′ of the human Eλ.-   21. The vertebrate or cell of any preceding aspect, wherein    optionally at least human JC gene segments Jλ1-Cλ1, Jλ2-Cλ2,    Jλ3-Cλ3, Jλ6-Cλ6 and Jλ7-Cλ7 are inserted in addition to the other    human gene segments.-   22. The vertebrate or cell of any preceding aspect, wherein    optionally the inserted human gene segments are in germline    configuration; optionally with the human inter-gene segment    sequences or the corresponding endogenous non-human vertebrate    inter-gene segment sequences.-   23. The vertebrate or cell of any preceding aspect, wherein    optionally an endogenous non-human vertebrate light chain enhancer    is maintained in the endogenous locus; optionally in germline    configuration. For example, when the endogenous locus is a kappa    locus, an endogenous kappa enhancer is maintained. This can be the    iEk and/or the 3′Ek, optionally in germline configuration with    respect to an endogenous light chain constant region. This may be    useful to help control of light chain expression in the non-human    vertebrate or cell.-   24. The vertebrate or cell of any preceding aspect, optionally    wherein the genome is heterozygous for the human lambda insertion at    the endogenous locus. For example, heterozygous for the human VJ or    VJC insertion at an endogenous kappa (eg, mouse or rat kappa) locus.    This aids and simplifies breeding of the vertebrates since the other    endogenous locus (eg, the other kappa locus) can be used to provide    a different transgenic Ig locus, such as a transgenic kappa locus    comprising human kappa V and J gene segments either upstream of the    endogenous mouse kappa constant region or upstream of a human kappa    constant region. In this case, the kappa enhancers (iEk and/or the    3′Ek) can be maintained in that kappa locus to aid expression in the    vertebrate by using endogenous control mechanisms.

In another embodiment, there is provided a non-human vertebrate or cellaccording to any preceding aspect, wherein

-   -   (a) the endogenous locus is an endogenous lambda locus (eg, in a        mouse), the genome being heterozygous for the insertion at the        lambda locus, thus one allele of the lambda locus comprising the        human Vλ and Jλ gene segment insertion (optionally with the        human CA gene segment insertion; optionally with the human EA        insertion) as described above;    -   (b) the other endogenous lambda allele comprises a plurality of        human VK gene segments and one or more human JK gene segments        upstream of a constant region (eg, a kappa constant region of        said non-human vertebrate species; a human kappa constant        region; the endogenous lambda constant region; or a human lambda        constant region); optionally with one or more kappa enhancers        (eg, iEk and/or the 3′Ek, eg, of said non-human vertebrate        species); and    -   (c) endogenous lambda and kappa chain expression has been        inactivated.

Thus, there is no expression of light chains comprising variable regionsderived from recombination of endogenous V and J regions, but there isexpression of human lambda and human kappa light chains from the allelesat the endogenous lambda locus. This is beneficial, since the designgreatly aids construction and breeding of vertebrates by avoiding needto provide transgenic loci at both the endogenous lambda and kappa loci.The endogenous kappa locus (and thus endogenous kappa chain expression)can be inactivated by inversion, deletion of kappa gene segments (eg,endogenous V and/or J and/or C kappa) and/or by insertion of aninterrupting sequence such as a marker (eg, neo) into the endogenouskappa locus.

The human kappa segment insertion into the endogenous lambda can becarried out, for example, by inserting a sequence corresponding to aportion of a human kappa locus comprising in germline configuration allfunctional human Vκ and Jκ (ie, optionally excluding pseudogenes andORFs; see the IMGT database); and optionally also a human iEκ.

-   25. The vertebrate or cell of aspect 24, optionally wherein the    genome comprises said human lambda gene segment insertion at one    endogenous non-human vertebrate kappa locus allele, and wherein the    other endogenous kappa locus allele comprises an insertion of human    kappa immunoglobulin V and J genes upstream of an endogenous    non-human vertebrate kappa constant region; optionally wherein an    endogenous kappa light chain enhancer is maintained in one or both    kappa locus; optionally in germline configuration.

The vertebrate or cell of aspect 24, optionally wherein the genomecomprises said human lambda gene segment insertion at one endogenousnon-human vertebrate lambda locus allele, and wherein the otherendogenous lambda locus allele comprises an insertion of human kappaimmunoglobulin V and J genes upstream of an endogenous non-humanvertebrate kappa constant region; optionally wherein an endogenouslambda light chain enhancer is maintained in one or both lambda locus;optionally in germline configuration.

-   26. The vertebrate or cell of claim 24, optionally wherein the    genome comprises said human lambda gene segment insertion at one    endogenous non-human vertebrate lambda locus allele, and wherein the    other endogenous lambda locus allele comprises an insertion of human    kappa immunoglobulin V and J genes upstream of an endogenous    non-human vertebrate kappa constant region; optionally wherein an    endogenous lambda light chain enhancer is maintained in one or both    kappa locus; optionally in germline configuration.-   27. The vertebrate or cell of any one of aspects 1 to 23, optionally    wherein the genome is homozygous for the human lambda insertion at    the endogenous non-human vertebrate locus.-   28. The vertebrate or cell of any one of aspects 1 to 23, optionally    wherein the genome is homozygous for a human lambda gene segment    insertion at the endogenous non-human vertebrate kappa and lambda    loci.-   29. The vertebrate or cell of any one of aspects 1 to 23 and 28,    optionally wherein the genome is homozygous for a human lambda gene    segment insertion at the endogenous non-human vertebrate lambda    loci, one endogenous kappa locus allele comprising a human lambda    gene segment insertion and the other endogenous kappa locus allele    comprising an insertion of a plurality of human Vκ and Jκ gene    segments upstream of a Cκ region for the expression of kappa light    chains comprising human kappa variable regions. Human kappa variable    regions are those derived from the recombination of human Vκ and JK.-   30. The vertebrate or cell of aspect 27 or 28, optionally wherein    the human lambda gene segment insertions at the kappa and lambda    loci are insertions of the same repertoire of human lambda gene    segments.-   31. The vertebrate or cell of aspect 27 or 28, optionally wherein    the human lambda gene segment insertions at the kappa loci are    different from the human lambda gene segment insertions at the    lambda loci. This is useful for expanding the potential repertoire    of variable regions for subsequent selection against antigen.-   32. A non-human vertebrate or a non-human vertebrate cell (eg, a    mouse, rat, mouse cell or a rat cell) whose genome comprises an Ig    gene segment repertoire produced by targeted insertion of human Ig    gene segments into one or more endogenous Ig loci, the genome    comprising the following light chain loci arrangement    -   (a) L at one endogenous kappa chain allele and K at the other        endogenous kappa chain allele; or    -   (b) L at one endogenous lambda chain allele and K at the other        endogenous lambda chain allele; or    -   (c) L at both endogenous kappa chain alleles;    -   (d) L at both endogenous lambda chain alleles;    -   (e) L at one endogenous kappa chain allele and the other        endogenous kappa chain allele has been inactivated; or    -   (f) L at one endogenous lambda chain allele and the other        endogenous lambda chain allele has been inactivated;    -   Wherein    -   L reperesents a human lambda gene segment insertion of at least        the functional human Vλ and Jλ (optionally also CA gene        segments) comprised by a human lambda chain Ig locus from Vλ3-1        to Cλ7 (eg, comprised by a human lambda chain Ig locus from 2-18        to Cλ7); and    -   K represents a human Vκ and Jκ insertion;    -   Wherein in the genome the human gene segments are inserted        upstream of a constant region for expression of light chains        comprising variable regions derived from the recombination of        human V and J gene segments.-   33. The vertebrate or cell according to aspect 32, optionally    wherein the genome comprises arrangement    -   (a) and L at one or both endogenous lambda chain alleles; or    -   (a) and K at one or both endogenous lambda chain alleles; or    -   (a) and L at one endogenous lambda chain allele and K at the        other endogenous lambda chain allele; or    -   (b) and L at one or both endogenous kappa chain alleles; or    -   (b) and K at one or both endogenous kappa chain alleles; or    -   (b) and L at one endogenous kappa chain allele and K at the        other endogenous kappa chain allele; or    -   (c) and K at one or both endogenous lambda chain alleles; or    -   (c) and L at one or both endogenous lambda chain alleles; or    -   (c) and L at one endogenous lambda chain allele and K at the        other endogenous lambda chain allele; or    -   (c) and both endogenous lambda chain alleles have been        inactivated; or    -   (d) and L at one or both endogenous kappa chain alleles; or    -   (d) and K at one or both endogenous kappa chain alleles; or    -   (d) and L at one endogenous kappa chain allele and K at the        other endogenous kappa chain allele; or    -   (d) and both endogenous kappa chain alleles have been        inactivated.-   34. The vertebrate or cell of aspect 32 or 33, optionally wherein    endogenous kappa chain expression is substantially or completely    inactivated. Endogenous kappa chains are kappa light chains    comprising variable regions derived from the recombination of    endogenous (non-human vertebrate) Vκ and Jκ gene segments.-   35. The vertebrate or cell of aspect 32, 33 or 34, optionally    wherein endogenous lambda chain expression is substantially or    completely inactive. Endogenous lambda chains are lambda light    chains comprising variable regions derived from the recombination of    endogenous (non-human vertebrate) Vλ and Jλ gene segments.-   36. The vertebrate or cell of any one of aspects 32 to 35,    optionally wherein each L insertion is upsteam of an endogenous    lambda or kappa constant region.-   37. The vertebrate or cell of any one of aspects 32 to 36,    optionally wherein each L insertion into a lambda locus is upsteam    of an endogenous lambda constant region.-   38. The vertebrate or cell of any one of aspects 32 to 36,    optionally wherein each L insertion into a kappa locus is upsteam of    an endogenous kappa constant region.-   39. The vertebrate or cell of any one of aspects 32 to 35,    optionally wherein each L insertion into a lambda locus is upsteam    of a human lambda constant region.-   40. The vertebrate or cell of any one of aspects 32 to 35,    optionally wherein each L insertion into a kappa locus is upsteam of    a human kappa constant region.-   41. The vertebrate or cell of any one of aspects 32 to 40,    optionally wherein each K insertion is upsteam of an endogenous    lambda or kappa constant region.-   42. The vertebrate or cell of any one of aspects 32 to 41,    optionally wherein each K insertion into a lambda locus is upsteam    of an endogenous lambda constant region.-   43. The vertebrate or cell of any one of aspects 32 to 42,    optionally wherein each K insertion into a kappa locus is upsteam of    an endogenous kappa constant region.-   44. The vertebrate or cell of any one of aspects 32 to 40,    optionally wherein each K insertion into a lambda locus is upsteam    of a human lambda constant region.-   45. The vertebrate or cell of any one of aspects 32 to 40 and 44,    optionally wherein each K insertion into a kappa locus is upsteam of    a human kappa constant region.-   46. The vertebrate or cell of any one of aspects 32 to 45,    optionally wherein the insertions are according to any one of    aspects 1 to 9, 11 to 16 and 20 to 31.-   47. The vertebrate or cell of any one of aspects 32 to 46,    optionally wherein each human lambda insertion is according to any    one of aspects 1 to 9, 11 to 16 and 20 to 31.-   48. The vertebrate or cell of any one of aspects 32 to 47,    optionally wherein each human kappa insertion is according to any    one of aspects 1 to 9, 11 to 16 and 20 to 31.-   49. The vertebrate or cell of any one of aspects 32 to 48,    optionally wherein each human lambda insertion comprises the    repertoire of human Vλ and Jλ (and optionally Cλ) gene segments.-   50. The vertebrate or cell of any one of aspects 32 to 48,    optionally wherein first and second (and optionally third) human    lambda insertions are made and the insertions comprise different    repertoires of human Vλ and Jλ (and optionally Cλ) gene segments.-   51. The vertebrate or cell of any one of aspects 32 to 50,    optionally wherein each human kappa insertion comprises the    repertoire of human Vκ and Jκ (and optionally Cκ) gene segments.-   52. The vertebrate or cell of any one of aspects 32 to 50,    optionally wherein first and second (and optionally third) human    kappa insertions are made and the insertions comprise different    repertoires of human Vκ and Jκ (and optionally Cκ) gene segments.-   53. The vertebrate or cell of any preceding aspect, optionally    wherein the genome comprises an immunoglobulin heavy chain locus    comprising human VH gene segments, eg, a heavy chain locus as herein    described which comprises human V, D and J gene segments.-   54. A method for producing an antibody or light chain comprising a    lambda variable region specific to a desired antigen, the method    comprising immunizing a vertebrate according to any preceding aspect    with the desired antigen and recovering the antibody or light chain    or recovering a cell producing the antibody or light chain.-   55. A method for producing a fully humanised antibody or antibody    light chain comprising carrying out the method of aspect 54 to    obtain an antibody or light chain comprising a lambda chain    non-human vertebrate constant region, and replacing the non-human    vertebrate constant region with a human constant region, optionally    by engineering of the nucleic acid encoding the antibody or light    chain.-   56. A humanised antibody or antibody light chain produced according    to aspect 54 or a derivative thereof; optionally for use in    medicine.-   57. Use of a humanised antibody or chain produced according to    aspect 54 or a derivative thereof in medicine.-   58. A method of inactivating endogenous Ig-VJ regions in the genome    of a non-human vertebrate or a non-human vertebrate cell (eg, a    mouse, rat, mouse cell or a rat cell), wherein the method comprises    inserting human immunoglobulin gene segments (eg, V and J gene    segments) in the genome between the endogenous Ig-VJ and an    endogenous enhancer or endogenous constant region to move the    endogenous Ig-VJ away from the enhancer or constant region, thereby    inactivating endogenous Ig-VJ regions.

In one embodiment, the endogenous Ig-VJ are heavy chain gene segments,the enhancer is an endogenous heavy chain enhancer, the constant regionis an endogenous heavy chain constant region and the human Ig genesegments comprise human VH, DH and JH gene segments.

In one embodiment, the endogenous Ig-VJ are lambda light chain genesegments, the enhancer is an endogenous lambda chain enhancer, theconstant region is an endogenous lambda chain constant region and thehuman Ig gene segments comprise human Vλ and Jλ gene segments.

In one embodiment, the endogenous Ig-VJ are kappa light chain genesegments, the enhancer is an endogenous kappa chain enhancer, theconstant region is an endogenous kappa chain constant region and thehuman Ig gene segments comprise human Vκ and Jκ gene segments.

A method of inactivating endogenous IgK-VJ regions in the genome of anon-human vertebrate or a non-human vertebrate cell (eg, a mouse, rat,mouse cell or a rat cell), wherein the method comprises inserting humanimmunoglobulin gene segments in the genome between the endogenous IgK-VJand Eκ enhancer to move the IgK-VJ away from the Eκ enhancer, therebyinactivating endogenous IgK-VJ regions.

-   59. The method of aspect 58, wherein optionally the human gene    segments comprise human VL and JL gene segments; optionally wherein    the insertion is an insertion as recited in any one of aspects 1 to    9, 11 to 16 and 20 to 31 or an insertion of human Vκ and Jκ gene    segments.-   60. A method of expressing immunoglobulin light chains in a    non-human vertebrate (eg, a mouse or rat), the light chains    comprising lambda variable regions (lambda light chains), wherein at    least 70 or 80% (for example, at least 70, 75, 80, 84, 85, 90, 95,    96, 97, 98 or 99%) of the variable regions of the lambda light    chains expressed by the vertebrate are derived from recombination of    human Vλ and Jλ gene segments, the method comprising providing in    the genome of the vertebrate an Ig gene segment repertoire produced    by targeted insertion of human Ig gene segments into one or more    endogenous Ig loci, the genome comprising human Vλ and Jλ gene    segments upstream of a constant region, wherein the method comprises    inserting at least the functional human Vλ and Jλ (optionally also    human Cλ) gene segments (and optionally inter-gene segment    sequences) comprised by a human lambda chain Ig locus from Vλ2-18 to    Cλ7 into an endogenous light chain locus of the vertebrate, wherein    at least 70 or 80% (for example, at least 70, 75, 80, 84, 85, 90,    95, 96, 97, 98 or 99%) of the variable regions of the lambda light    chains expressed by the vertebrate are derived from recombination of    human Vλ and Jλ gene segments; the method comprising expressing said    light chains in the vertebrate and optionally isolating one or more    of said light chains (eg, as part of a 4-chain antibody).

In one embodiment, the method further comprises isolating from thevertebrate a lambda light chain comprising a variable region derivedfrom recombination of human Vλ and Jλ gene segments. In an example, themethod comprises immunising the mouse with an antigen (eg, a humanantigen) prior to isolating the lambda light chain. In an example, thelight chain is part of an antibody, eg, an antibody that specificallybinds the antigen.

In one embodiment, the use further comprises isolating splenic tissue(eg, the spleen) from the mouse; optionally followed by isolating atleast one antigen-specific B-cell from the tissue, wherein the B-cell(s)expresses said lambda light chain. For example, said lambda light chainis provided by an antibody that specifically binds a predeterminedantigen (eg, a human antigen). In one example, the use comprisesimmunising the mouse with the antigen (eg, a human antigen) prior toisolating the splenic tissue or lambda light chain. In an example, theuse comprises isolating the lambda light chain produced by the B-cell(or by a hybridoma produced by fusion of the B-cell with a myelomacell). In an example, the use comprises making a hybridoma from a B-cellisolated from the splenic tissue, wherein the hybridoma expresses saidlambda light chain or a derivative thereof. Optionally, the usecomprises making a derivative of the isolated antibody or lambda lightchain. Examples of derivative antibodies (according to any aspectherein) are antibodies that have one or more mutations compared to theisolated antibody (eg, to improve antigen-binding affinity and/or toenhance or inactivate Fc function) Such mutants specifically bind theantigen. Mutation or adaptation to produce a derivative includes, eg,mutation to produce Fc enhancement or inactivation. A derivative can bean antibody following conjugation to a toxic payload or reporter orlabel or other active moiety. In another example, a chimaeric antibodychain or antibody isolated from a cell of vertebrate of the invention ismodified by replacing one or all human constant regions thereof by acorresponding human constant region. For example, all constant regionsof an antibody isolated from such a cell or vertebrate are replaced withhuman constant regions to produce a fully human antibody (ie, comprisinghuman variable and constant regions). Such an antibody is useful foradministration to human patients to reduce anti-antibody reaction by thepatient.

-   61. A method of expressing immunoglobulin light chains in a    non-human vertebrate (eg, a mouse or rat), wherein at least 60% (for    example, at least 65, 70, 80, 84, 85, 90, 95, 96, 97, 98 or 99%) of    the light chains expressed by the vertebrate are provided by human    lambda light chains, the method comprising providing in the genome    of the vertebrate an Ig gene segment repertoire produced by targeted    insertion of human Ig gene segments into one or more endogenous Ig    loci, the genome comprising (i) human Vλ and Jλ gene segments    upstream of a constant region, wherein the human Vλ and Jλ gene    segments are provided by inserting at least the functional human Vλ    and Jλ (optionally also human Cλ) gene segments (and optionally    inter-gene segment sequences) comprised by a human lambda chain Ig    locus from Vλ2-18 to Cλ7 into an endogenous light chain locus of the    vertebrate and (ii) kappa V gene segments upstream of a constant    region, wherein the vertebrate expresses immunoglobulin light chains    comprising human lambda variable regions (human lambda light chains)    and at least 60% (for example, greater than 65, 70, 80, 84, 85, 90,    95, 96, 97, 98 or 99%) of the light chains expressed by the    vertebrate are provided by said human lambda light chains; the    method comprising expressing said light chains in the vertebrate and    optionally isolating one or more of said light chains (eg, as part    of a 4-chain antibody).

In one embodiment, the method further comprises isolating from thevertebrate a lambda light chain comprising a variable region derivedfrom recombination of human Vλ and Jλ gene segments. In an example, themethod comprises immunising the mouse with an antigen (eg, a humanantigen) prior to isolating the lambda light chain. In an example, thelight chain is part of an antibody, eg, an antibody that specificallybinds the antigen.

In one embodiment, the use further comprises isolating splenic tissue(eg, the spleen) from the mouse; optionally followed by isolating atleast one antigen-specific B-cell from the tissue, wherein the B-cell(s)expresses said lambda light chain. For example, said lambda light chainis provided by an antibody that specifically binds a predeterminedantigen (eg, a human antigen). In one example, the use comprisesimmunising the mouse with the antigen (eg, a human antigen) prior toisolating the splenic tissue or lambda light chain. In an example, theuse comprises isolating the lambda light chain produced by the B-cell(or by a hybridoma produced by fusion of the B-cell with a myelomacell). In an example, the use comprises making a hybridoma from a B-cellisolated from the splenic tissue, wherein the hybridoma expresses saidlambda light chain or a derivative thereof. Optionally, the usecomprises making a derivative of the isolated antibody or lambda lightchain. Examples of derivative antibodies (according to any aspectherein) are antibodies that have one or more mutations compared to theisolated antibody (eg, to improve antigen-binding affinity and/or toenhance or inactivate Fc function) Such mutants specifically bind theantigen.

-   62. A method of expressing human immunoglobulin VJC light chains in    a non-human vertebrate (eg, a mouse or rat), the method comprising    providing in the genome of the vertebrate an Ig gene segment    repertoire produced by targeted insertion of human Ig gene segments    into one or more endogenous Ig loci, wherein the method comprises    inserting at least the functional human Vλ, Jλ and CA gene segments    (and optionally inter-gene segment sequences) comprised by a human    lambda chain Ig locus from Vλ3-1 to Cλ7 (eg, comprised by a human    lambda chain Ig locus from 2-18 to Cλ7) into an endogenous non-human    vertebrate kappa light chain locus upstream of an endogenous    non-human vertebrate kappa constant region for expression of a human    VJC light chain; the method comprising expressing said light chains    in the vertebrate and optionally isolating one or more of said light    chains (eg, as part of a 4-chain antibody).

In one embodiment, the method further comprises isolating from thevertebrate a lambda light chain comprising a variable region derivedfrom recombination of human Vλ and Jλ gene segments. In an example, themethod comprises immunising the mouse with an antigen (eg, a humanantigen) prior to isolating the lambda light chain. In an example, thelight chain is part of an antibody, eg, an antibody that specificallybinds the antigen.

In one embodiment, the use further comprises isolating splenic tissue(eg, the spleen) from the mouse; optionally followed by isolating atleast one antigen-specific B-cell from the tissue, wherein the B-cell(s)expresses said lambda light chain. For example, said lambda light chainis provided by an antibody that specifically binds a predeterminedantigen (eg, a human antigen). In one example, the use comprisesimmunising the mouse with the antigen (eg, a human antigen) prior toisolating the splenic tissue or lambda light chain. In an example, theuse comprises isolating the lambda light chain produced by the B-cell(or by a hybridoma produced by fusion of the B-cell with a myelomacell). In an example, the use comprises making a hybridoma from a B-cellisolated from the splenic tissue, wherein the hybridoma expresses saidlambda light chain or a derivative thereof. Optionally, the usecomprises making a derivative of the isolated antibody or lambda lightchain. Examples of derivative antibodies (according to any aspectherein) are antibodies that have one or more mutations compared to theisolated antibody (eg, to improve antigen-binding affinity and/or toenhance or inactivate Fc function) Such mutants specifically bind theantigen.

-   63. The method of any one of aspects 38 to 40, optionally wherein    the vertebrate is according to any one of the other aspects.-   64. An antibody light chain isolated according to the method of any    one of aspects 58 to 63 or a derivative thereof, or an antibody    comprising such a light chain or derivative; optionally for use in    medicine.-   65. Use of an antibody light chain isolated according to the method    of any one of aspects 58 to 63 or a derivative thereof (or an    antibody comprising such a light chain or derivative) in medicine.-   66. A non-human vertebrate (eg, a mouse or rat) according to any one    of aspects 1 to 53 for expressing light chains comprising lambda    variable regions (lambda light chains), wherein at least 70 or 80%    (for example, at least 70, 75, 80, 84, 85, 90, 95, 96, 97, 98 or 99%    or 100%) of the variable regions of the lambda light chains    expressed by the vertebrate are derived from recombination of human    Vλ and Jλ gene segments.

A non-human vertebrate (eg, a mouse or rat) according to any one ofaspects 1 to 53 expressing light chains comprising lambda variableregions (lambda light chains), wherein at least 70 or 80% (for example,at least 70, 75, 80, 84, 85, 90, 95, 96, 97, 98 or 99% or 100%) of thevariable regions of the lambda light chains expressed by the vertebrateare derived from recombination of human Vλ and Jλ gene segments.

-   67. A non-human vertebrate (eg, a mouse or rat) according to any one    of aspects 1 to 53 for expressing light chains, wherein at least 60%    (for example, greater than 65, 70, 80, 84, 85, 90, 95, 96, 97, 98 or    99% or 100%) of the light chains expressed by the vertebrate are    provided by human lambda light chains.

A non-human vertebrate (eg, a mouse or rat) according to any one ofaspects 1 to 53 expressing light chains, wherein at least 60% (forexample, greater than 65, 70, 80, 84, 85, 90, 95, 96, 97, 98 or 99% or100%) of the light chains expressed by the vertebrate are provided byhuman lambda light chains.

-   68. A non-human vertebrate (eg, a mouse or rat) according to aspect    7 for expressing light chains comprising lambda variable regions    (lambda light chains), wherein expression of lambda light chains    comprising human lambda variable regions dominates over expression    of lambda light chains comprising endogenous non-human vertebrate    lambda variable regions: and optionally for inactivating expression    of endogenous non-human vertebrate lambda variable regions from the    endogenous light chain locus.

A non-human vertebrate (eg, a mouse or rat) according to aspect 7expressing light chains comprising lambda variable regions (lambda lightchains), wherein expression of lambda light chains comprising humanlambda variable regions dominates over expression of lambda light chainscomprising endogenous non-human vertebrate lambda variable regions: andoptionally for inactivating expression of endogenous non-humanvertebrate lambda variable regions from the endogenous light chainlocus.

-   69. A non-human vertebrate (eg, a mouse or rat) according to aspect    7, 8, 9 or 10 for inactivating expression of endogenous non-human    vertebrate lambda variable regions from the endogenous light chain    locus.

The percentage expression or level of expression of antibody chains canbe determined at the level of light chain mRNA transcripts in B-cells(eg, peripheral blood lymphocytes). Alternatively or additionally, thepercentage expression is determined at the level of antibody lightchains in serum or blood of the vertebrates. Additionally oralternatively, the expression can be determined by FACS (fluorescenceactivated cell sorting) analysis of B cells. For example, by assessingmouse C kappa or human C lambda expression on cell surface when thehuman lambda variable regions are expressed with mouse C kappa or humanC lambda regions respectively.

The term a “lambda light chain” in these aspects refers to a light chaincomprising a variable region sequence (at RNA or amino acid level)derived from the recombination of Vλ and Jλ gene segments. Thus a “humanlambda variable region”, for example, is a variable region derived fromthe recombination of human Vλ and Jλ gene segments. The constant regioncan be a kappa or lambda constant region, eg, a human or mouse constantregion.

The vertebrate in these aspects is, for example naïve (ie, not immunisedwith a predetermined antigen, as the term is understood in the art; forexample, such a vertebrate that has been kept in a relatively sterileenvironment as provided by an animal house used for R&D). In anotherexample, the vertebrate has been immunised with a predetermined antigen,eg, an antigen bearing a human epitope.

Reference to “functional” human gene segments acknowledges that in ahuman Ig lambda locus some V gene segments are non-functionalpseudogenes (eg, Vλ3-17, Vλ3-15, Vλ3-13, Vλ3-7, Vλ3-6, Vλ2-5, Vλ3-4,Vλ3-2; see the IMGT database: at World Wide Web (www)imgt.org/IMGTrepertoire/index.php?section=LocusGenes&repertoire=locus&species=human&group=IGL. Also, Jλ4-Cλ4 and Jλ5-Cλ5 are not functional in humans.The term “functional” when referring to gene segments excludespseudogenes. An example of functional human Vλ gene segments is thegroup Vλ2-18, Vλ3-16, V2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3and Vλ3-1. An example of functional human Jλ gene segments is the groupJλ1, Jλ2 and Jλ3; or Jλ1, Jλ2 and Jλ7; or Jλ2, Jλ3 and Jλ7; or Jλ1, Jλ2,Jλ3 and Jλ7. An example of functional human CA gene segments is thegroup Cλ1, Cλ2 and Cλ3; or Cλ1, Cλ2 and Cλ7; or Cλ2, Cλ3 and Cλ7; orCλ1, Cλ2, Cλ3 and Cλ7.

In one embodiment, the lambda light chains, together with heavy chainsexpressed in the cells or vertebrates of the invention, form antibodies.The heavy chains can be expressed from a transgenic heavy chain locus asherein described. For example the genome of the cell or vertebratecomprises a heavy chain locus in which is a chimaeric immunoglobulinheavy chain locus comprising one or more human V gene segments, one ormore human D gene segments and one or more human J gene segmentsupstream of a mu constant region of said non-human species; endogenousheavy chain expression has been substantially inactivated; and the heavychain locus comprises an Ep enhancer of said non-human vertebratespecies.

In one embodiment of the vertebrate or cell, all endogenous enhancersare deleted from the endogenous locus in which the human gene segmentsare inserted. Thus, when a human enhancer (eg, Eλ) is inserted, thiscontrols the transgenic locus in the absence of the effect of other,endogenous, enhancers (for example, kappa enhancers if the locus is anendogenous kappa enhancer). This may be useful to avoid non-humanvertebrate-like kappa:lambda expression ratios (eg, to steer expressionto a higher ratio of lambda:kappa in mice).

When endogenous light chain (eg, kappa or lambda) expression issubstantially inactive or inactivated as described herein, less than 10,5, 4, 3, 2, 1 or 0.5% of such endogenous light chains are expressed orexpressible. In one example, there is complete inactivation so no suchlight chains are expressed or expressible.

Optionally the vertebrate of the invention is naïve. Thus, thevertebrate has not been immunised with a predetermined antigen.

Where, for example, a cell of the invention is an ES cell or other IPSstem cell or other pluripotent stem cell, the cell can develop into avertebrate of the invention. For example, the cell can be implanted intoa blastocyst from a foster mother and developed into an embryo andanimal according to standard techniques.

In one embodiment, where human kappa gene segments are inserted, eachinsertion comprises human kappa gene segments

-   -   (i) VK1-5, VK1-6, VK1-8 and VK1-9 (and optionally VK5-2 and        VK4-1); or    -   (ii) VK1-5, VK1-6, VK1-8, VK1-9, VK3-11, VK1-12, VK3-15, VK1-16,        VK1-17, VK3-20 (and optionally Vκ 2-24 and/or VK1-13); or    -   (iii) VK1-5, VK1-6, VK1-8, VK1-9, VK3-11, VK1-12, VK3-15,        VK1-16, VK1-17, VK3-20, Vκ 2-24, VK1-27, VK2-28, VK2-30 and        VK1-33 (and optionally Vκ 2-29 and/or VK2-40 and/or VK1-39);    -   and optionally    -   (iv) JK1, JK2, JK3, JK4 and JK5.

In one embodiment, the human kappa insertion also comprises a human iEκand/or human 3′Eκ downstream of the human J gene segments in the locus.

Transgenic Mice of the Invention Expressing Essentially ExclusivelyHuman Heavy Chain Variable Regions Develop Normal Splenic and BMCompartments & Normal Ig Expression In Which the Ig Comprise Human HeavyChain Variable Regions

The present inventors surprisingly observed normal Ig subtype expression& B-cell development in transgenic mice of the invention expressingantibodies with human heavy chain variable regions substantially in theabsence of endogenous heavy and kappa chain expression. See Example 16below.

The inventors observed that surprisingly the inactivation of endogenousheavy chain variable region expression in the presence of human variableregion expression does not change the ratio of B-cells in the spleniccompartment (FIG. 66) or bone marrow B progenitor compartment (FIG. 67)and the immunoglobulin levels in serum are normal and the correct Igsubtypes are expressed (FIG. 68). These data demonstrate that insertedhuman heavy chain gene segments according to the invention (eg, aninsertion of at least human V_(H) gene segments V_(H)2-5, 7-4-1, 4-4,1-3, 1-2, 6-1, and all the human D and JH gene segments D1-1, 2-2, 3-3,4-4, 5-5, 6-6, 1-7, 2-8, 3-9, 5-12, 6-13, 2-15, 3-16, 4-17, 6-19, 1-20,2-21, 3-22, 6-25, 1-26 and 7-27; and J1, J2, J3, J4, J5 and J6,optionally the alleles of Table 7) are fully functional for VDJ genesegment rearrangement from the transgenic heavy chain locus, B-cellreceptor (BCR) signalling and proper B-cell maturation

The invention therefore provides the following aspects (numberingstarting at aspect 70):—

-   70. A mouse that expresses or for expressing immunoglobulin heavy    chains comprising human variable regions, wherein the heavy chains    expressed by the mouse are essentially exclusively said heavy chains    comprising human variable regions; and said heavy chains comprising    human variable regions are expressed as part of serum IgG1, IgG2b    and IgM (and optionally IgG2a) antibodies in the mouse;

the mouse comprising an immunoglobulin heavy chain locus comprisinghuman VH, DH and JH gene segments upstream of a mouse constant region(eg, C-mu and/or C-delta and/or C-gamma; such as (in a 5′ to 3′orientation) mouse C-mu and mouse C-delta and mouse C-gamma), wherein

-   -   (a) the mouse is capable of expressing immunoglobulin heavy        chains comprising human variable regions and the heavy chains        expressed by the mouse are essentially exclusively said heavy        chains comprising human variable regions; and    -   (b) the mouse expresses serum IgG1, IgG2b and IgM (and        optionally IgG2a) antibodies comprising said heavy chains.

Ig isotypes can be determined, for example, using isotype-matched toolantibodies as will be readily familiar to the skilled person (and asillustrated in Example 16).

In an embodiment, the mouse is naïve.

-   71. The mouse of aspect 70 for expressing a normal relative    proportion of serum IgG1, IgG2a, IgG2b and IgM antibodies.

By “normal” is meant comparable to expression in a mouse (eg, a naïvemouse) expressing only mouse antibody chains, eg, a mouse whose genomecomprises only wild-type functional Ig heavy and light chain loci, eg, awild-type mouse.

-   72. The mouse of aspect 70 or 71, wherein the mouse expresses a    normal relative proportion of serum IgG1, IgG2a, IgG2b and IgM    antibodies.

By “normal” is meant comparable to expression in a mouse (eg, a naïvemouse) expressing only mouse antibody chains, eg, a mouse whose genomecomprises only wild-type functional Ig heavy and light chain loci, eg, awild-type mouse.

-   73. The mouse of any one of aspects 70 to 72, for expressing in the    mouse    -   (i) serum IgG1 at a concentration of about 25-350 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-200 μg/ml;    -   (iii) serum IgG2b at a concentration of about 30-800 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-300 μg/ml;    -   or    -   (i) serum IgG1 at a concentration of about 10-600 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-500 μg/ml;    -   (iii) serum IgG2b at a concentration of about 20-700 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-700 μg/ml;    -   as determined by Ig capture on a plate followed by incubation        (eg, for one hour at RT, eg, for one hour at 20° C.) with        anti-mouse isotype-specific labelled antibodies and        quantification of Ig using the label (eg, using anti-mouse Ig        isotype specific antibodies each conjugated to horseradish        peroxidase conjugated at a ratio of 1/10000 in PBS with 0.1%        Tween™, followed by development of the label with        tetramethylbenzidine substrate (TMB) for 4-5 minutes in the dark        at room temperature (eg, 20° C.), adding sulfuric acid to stop        development of the label and reading of the label at 450 nm).

For example, the mouse of any one of aspects 70 to 72, for expressing inthe mouse serum IgG1 at a concentration of about 25-150 μg/ml;

-   -   (ii) serum IgG2a at a concentration of about 0-200 μg/ml;    -   (iii) serum IgG2b at a concentration of about 30-300 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-200 μg/ml;    -   or    -   (i) serum IgG1 at a concentration of about 10-200 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-500 μg/ml;    -   (iii) serum IgG2b at a concentration of about 20-400 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-700 μg/ml;    -   as determined by Ig capture on a plate followed by incubation        (eg, for one hour at RT, eg, for one hour at 20° C.) with        anti-mouse isotype-specific labelled antibodies and        quantification of Ig using the label (eg, using anti-mouse Ig        isotype specific antibodies each conjugated to horseradish        peroxidase conjugated at a ratio of 1/10000 in PBS with 0.1%        Tween™, followed by development of the label with        tetramethylbenzidine substrate (TMB) for 4-5 minutes in the dark        at room temperature (eg, 20° C.), adding sulfuric acid to stop        development of the label and reading of the label at 450 nm).

The mouse of any one of aspects 70 to 72, for expressing in the mouse Igin the relative proportions of serum IgG1 at a concentration of about25-350 μg/ml;

-   -   (ii) serum IgG2a at a concentration of about 0-200 μg/ml;    -   (iii) serum IgG2b at a concentration of about 30-800 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-300 μg/ml;    -   or    -   (i) serum IgG1 at a concentration of about 10-600 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-500 μg/ml;    -   (iii) serum IgG2b at a concentration of about 20-700 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-700 μg/ml;    -   as determined by Ig capture on a plate followed by incubation        (eg, for one hour at RT, eg, for one hour at 20° C.) with        anti-mouse isotype-specific labelled antibodies and        quantification of Ig using the label (eg, using anti-mouse Ig        isotype specific antibodies each conjugated to horseradish        peroxidase conjugated at a ratio of 1/10000 in PBS with 0.1%        Tween™, followed by development of the label with        tetramethylbenzidine substrate (TMB) for 4-5 minutes in the dark        at room temperature (eg, 20° C.), adding sulfuric acid to stop        development of the label and reading of the label at 450 nm).

For example, the mouse of any one of aspects 70 to 72, for expressing inthe mouse Ig in the relative proportions of

-   -   (i) serum IgG1 at a concentration of about 25-150 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-200 μg/ml;    -   (iii) serum IgG2b at a concentration of about 30-300 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-200 μg/ml;    -   or serum IgG1 at a concentration of about 10-200 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-500 μg/ml;    -   (iii) serum IgG2b at a concentration of about 20-400 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-700 μg/ml;    -   as determined by Ig capture on a plate followed by incubation        (eg, for one hour at RT, eg, for one hour at 20° C.) with        anti-mouse isotype-specific labelled antibodies and        quantification of Ig using the label (eg, using anti-mouse Ig        isotype specific antibodies each conjugated to horseradish        peroxidase conjugated at a ratio of 1/10000 in PBS with 0.1%        Tween™, followed by development of the label with        tetramethylbenzidine substrate (TMB) for 4-5 minutes in the dark        at room temperature (eg, 20° C.), adding sulfuric acid to stop        development of the label and reading of the label at 450 nm).

-   74. The mouse of any one of aspects 70 to 73, wherein the mouse    expresses    -   (i) serum IgG1 at a concentration of about 25-350 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-200 μg/ml;    -   (iii) serum IgG2b at a concentration of about 30-800 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-300 μg/ml;    -   or    -   (i) serum IgG1 at a concentration of about 10-600 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-500 μg/ml;    -   (iii) serum IgG2b at a concentration of about 20-700 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-700 μg/ml;    -   as determined by Ig capture on a plate followed by incubation        (eg, for one hour at RT, eg, for one hour at 20° C.) with        anti-mouse isotype-specific labelled antibodies and        quantification of Ig using the label (eg, using anti-mouse Ig        isotype specific antibodies each conjugated to horseradish        peroxidase conjugated at a ratio of 1/10000 in PBS with 0.1%        Tween™, followed by development of the label with        tetramethylbenzidine substrate (TMB) for 4-5 minutes in the dark        at room temperature (eg, 20° C.), adding sulfuric acid to stop        development of the label and reading of the label at 450 nm).

For example, the mouse of any one of aspects 70 to 72, the mouseexpresses

-   -   (i) serum IgG1 at a concentration of about 25-150 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-200 μg/ml;    -   (iii) serum IgG2b at a concentration of about 30-300 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-200 μg/ml;    -   or    -   (i) serum IgG1 at a concentration of about 10-200 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-500 μg/ml;    -   (iii) serum IgG2b at a concentration of about 20-400 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-700 μg/ml;    -   as determined by Ig capture on a plate followed by incubation        (eg, for one hour at RT, eg, for one hour at 20° C.) with        anti-mouse isotype-specific labelled antibodies and        quantification of Ig using the label (eg, using anti-mouse Ig        isotype specific antibodies each conjugated to horseradish        peroxidase conjugated at a ratio of 1/10000 in PBS with 0.1%        Tween™, followed by development of the label with        tetramethylbenzidine substrate (TMB) for 4-5 minutes in the dark        at room temperature (eg, 20° C.), adding sulfuric acid to stop        development of the label and reading of the label at 450 nm).

The mouse of any one of aspects 70 to 73, wherein the mouse expresses Igin the relative proportions of

-   -   (i) serum IgG1 at a concentration of about 25-350 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-200 μg/ml;    -   (iii) serum IgG2b at a concentration of about 30-800 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-300 μg/ml;    -   or    -   (i) serum IgG1 at a concentration of about 10-600 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-500 μg/ml;    -   (iii) serum IgG2b at a concentration of about 20-700 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-700 μg/ml;    -   as determined by Ig capture on a plate followed by incubation        (eg, for one hour at RT, eg, for one hour at 20° C.) with        anti-mouse isotype-specific labelled antibodies and        quantification of Ig using the label (eg, using anti-mouse Ig        isotype specific antibodies each conjugated to horseradish        peroxidase conjugated at a ratio of 1/10000 in PBS with 0.1%        Tween™, followed by development of the label with        tetramethylbenzidine substrate (TMB) for 4-5 minutes in the dark        at room temperature (eg, 20° C.), adding sulfuric acid to stop        development of the label and reading of the label at 450 nm).

For example, the mouse of any one of aspects 70 to 72, the mouseexpresses Ig in the relative proportions of

-   -   (i) serum IgG1 at a concentration of about 25-150 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-200 μg/ml;    -   (iii) serum IgG2b at a concentration of about 30-300 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-200 μg/ml;    -   or    -   (i) serum IgM at a concentration of about 10-200 μg/ml    -   (ii) serum IgG2a at a concentration of about 0-500 μg/ml;    -   (iii) serum IgG2b at a concentration of about 20-400 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-700 μg/ml;    -   as determined by Ig capture on a plate followed by incubation        (eg, for one hour at RT, eg, for one hour at 20° C.) with        anti-mouse isotype-specific labelled antibodies and        quantification of Ig using the label (eg, using anti-mouse Ig        isotype specific antibodies each conjugated to horseradish        peroxidase conjugated at a ratio of 1/10000 in PBS with 0.1%        Tween™, followed by development of the label with        tetramethylbenzidine substrate (TMB) for 4-5 minutes in the dark        at room temperature (eg, 20° C.), adding sulfuric acid to stop        development of the label and reading of the label at 450 nm).

-   75. The mouse of any one of aspects 70 to 74 for expressing said    heavy chains from splenic B-cells in a mouse that produces a normal    proportion or percentage of mature splenic B-cells, eg as determined    by FACS.    -   By “normal” is meant comparable to mature splenic B-cell        production in a mouse (eg, a naïve mouse) expressing only mouse        antibody chains, eg, a mouse whose genome comprises only        wild-type functional Ig heavy and light chain loci, eg, a        wild-type mouse.    -   For example, at least 40, 50, 60 or 70% of total splenic B-cells        produced by the mouse of the invention are mature B-cells.        Splenic B-cells are B220+ and express B220 at relatively high        levels as the skilled person will know. Mature splenic B-cells        express B220 and IgD, both at relatively high levels as will be        known by the skilled person. IgM expression is relatively low in        mature splenic B-cells, again as is known in the art. For        example, see J Exp Med. 1999 Jul. 5; 190(1):75-89; “B cell        development in the spleen takes place in discrete steps and is        determined by the quality of B cell receptor-derived signals”;        Loder F et al.

Optionally the mouse produces a normal ratio of T1, T2 and maturesplenic B-cells, eg, as determined by FACS. For example, the mouse ofthe invention produces about 40-70% mature splenic B-cells, 15-35%splenic T1 cells; and 5-10% splenic T2 cells (percentage with referenceto the total splenic B220-positive (high) population). For example,about 40-60% mature splenic B-cells, 15-30% splenic T1 cells; and 5-10%splenic T2 cells. By “normal” is meant comparable to a T1/T2/maturesplenic B-cell proportion in a mouse (eg, a naïve mouse) expressing onlymouse antibody chains, eg, a mouse whose genome comprises only wild-typefunctional Ig heavy and light chain loci, eg, a wild-type mouse.

-   76. The mouse of any one of aspects 70 to 75, wherein the mouse    produces a normal proportion or percentage of mature splenic    B-cells, eg as determined by FACS.-   77. A mouse that expresses or for expressing immunoglobulin heavy    chains comprising human variable regions, wherein the heavy chains    expressed by the mouse are essentially exclusively said heavy chains    comprising human variable regions and are expressed in a mouse that    produces a normal proportion or percentage of mature splenic B-cells    (eg, as determined by FACS); the mouse comprising an immunoglobulin    heavy chain locus comprising human VH, DH and JH gene segments    upstream of a mouse constant region (eg, C-mu and/or C-delta and/or    C-gamma; such as (in a 5′ to 3′ orientation) and wherein the mouse    produces a normal proportion or percentage of mature splenic    B-cells.

By “normal” is meant comparable to mature splenic B-cell production in amouse (eg, a naïve mouse) expressing only mouse antibody chains, eg, amouse whose genome comprises only wild-type functional Ig heavy andlight chain loci, eg, a wild-type mouse.

For example, at least 40, 50, 60 or 70% of total splenic B-cellsproduced by the mouse of the invention are mature B-cells. SplenicB-cells are B220+ and express B220 at relatively high levels as theskilled person will know. Mature splenic B-cells express B220 and IgD,both at relatively high levels as will be known by the skilled person.IgM expression is relatively low in mature splenic B-cells, again as isknown in the art. For example, see J Exp Med. 1999 Jul. 5; 190(1):75-89;“B cell development in the spleen takes place in discrete steps and isdetermined by the quality of B cell receptor-derived signals”; Loder Fet al.

Optionally the mouse produces a normal ratio of T1, T2 and maturesplenic B-cells, eg, as determined by FACS. For example, the mouse ofthe invention produces about 40-70% mature splenic B-cells, 15-35%splenic T1 cells; and 5-10% splenic T2 cells (percentage with referenceto the total splenic B220-positive (high) population). For example,about 40-60% mature splenic B-cells, 15-30% splenic T1 cells; and 5-10%splenic T2 cells. By “normal” is meant comparable to a T1/T2/maturesplenic B-cell proportion in a mouse (eg, a naïve mouse) expressing onlymouse antibody chains, eg, a mouse whose genome comprises only wild-typefunctional Ig heavy and light chain loci, eg, a wild-type mouse.

-   78. The mouse of any one of aspects 70 to 77 for expressing said    heavy chains in a mouse that produces a normal proportion or    percentage of bone marrow B-cell progenitor cells (eg as determined    by FACS).

In one embodiment, the mouse is for expressing said heavy chains in amouse that produces a normal proportion or percentage of bone marrowpre-, pro and prepro-B-cells (eg as determined by FACS). See J Exp Med.1991 May 1; 173(5):1213-25; “Resolution and characterization of pro-Band pre-pro-B cell stages in normal mouse bone marrow”; Hardy R R et alfor more discussion on progenitor cells.

By “normal” is meant comparable to bone marrow B-cell production in amouse (eg, a naïve mouse) expressing only mouse antibody chains, eg, amouse whose genome comprises only wild-type functional Ig heavy andlight chain loci, eg, a wild-type mouse.

-   79. The mouse of any one of aspects 70 to 78, wherein the mouse    produces a normal proportion or percentage of bone marrow B-cell    progenitor cells (eg, as determined by FACS).

In one embodiment, the mouse produces a normal proportion or percentageof bone marrow pre-, pro and prepro-B-cells (eg as determined by FACS).

By “normal” is meant comparable to bone marrow B-cell production in amouse (eg, a naïve mouse) expressing only mouse antibody chains, eg, amouse whose genome comprises only wild-type functional Ig heavy andlight chain loci, eg, a wild-type mouse.

-   80. A mouse that expresses or for expressing immunoglobulin heavy    chains comprising human variable regions, wherein the heavy chains    expressed by the mouse are essentially exclusively said heavy chains    comprising human variable regions and are expressed in a mouse that    produces a normal proportion or percentage of bone marrow B-cell    progenitor cells (eg, as determined by FACS), the mouse comprising    an immunoglobulin heavy chain locus comprising human VH, DH and JH    gene segments upstream of a mouse constant region (eg, C-mu and/or    C-delta and/or C-gamma; such as (in a 5′ to 3′ orientation) and    wherein the mouse produces a normal proportion or percentage of bone    marrow B-cell progenitor cells.

In one embodiment, the mouse is for expressing said heavy chains in amouse that produces a normal proportion or percentage of bone marrowpre-, pro and prepro-B-cells (eg as determined by FACS).

By “normal” is meant comparable to bone marrow B-cell production in amouse (eg, a naïve mouse) expressing only mouse antibody chains, eg, amouse whose genome comprises only wild-type functional Ig heavy andlight chain loci, eg, a wild-type mouse.

-   81. The mouse of any one of aspects 70 to 80, wherein at least 90%    of the heavy chains are heavy chains comprising human variable    regions.

For example, at least 90, 95, 96, 97, 98, 99 or 99.5% or 100% of theheavy chains comprise human variable regions, ie, variable regionsderived from the recombination of human VH with human D and JH genesegments.

-   82. The mouse of any one of aspects 70 to 81, wherein the mouse    constant region comprises a mouse C-mu region, a C-delta region and    a C-gamma region.

In one embodiment, each of the C regions is an endogenous, mouseC-region. In one embodiment at least the C-mu and the C-delta regionsare mouse C regions. This is useful for harnessing the endogenouscontrol mechanisms involved in the development of the various B-celltypes and progenitors in the spleen and bone marrow.

In one embodiment, the C-gamma region is a human C-gamma region. This isbeneficial for producing class-switched gamma-type heavy chains in themouse in which essentially all of the expressed heavy chains have humanvariable regions and human constant regions.

-   83. The mouse of any one of aspects 70 to 82, wherein there is a    mouse heavy chain enhancer between the human gene segments and the    mouse constant region. This is useful for harnessing the endogenous    mouse antibody- and B-cell development control mechanisms.-   84. The mouse of any one of aspects 70 to 83, wherein there is a    mouse S-mu switch between the human gene segments and the mouse    constant region.-   85. The mouse of any one of aspects 70 to 84, wherein the genome of    the mouse comprises endogenous mouse heavy chain locus V, D and J    gene segments upstream of the human gene segments.-   86. The mouse of aspect 85, wherein the mouse V, D and J gene    segments are present together with the endogenous inter-gene segment    sequences.-   87. The mouse of aspect 85 or 86, wherein the mouse gene segments    are in inverted orientation. Thus, they are inverted with respect to    the wild-type orientation in a mouse genome. They are thus inverted    relative to the orientation of the mouse constant region.-   88. The mouse of any one of aspects 70 to 87, wherein the mouse    expresses light chains comprising human variable regions (eg, kappa    light chains comprising human kappa variable regions). Thus, the    human variable regions are derived from the recombination of human    VL and JL gene segments, eg, human Vκ and human JK.-   89. The mouse of aspect 88, comprising human Vκ and Jκ gene segments    upstream of a mouse CL (eg, endogenous CO; optionally wherein the    human Vκ and Jκ gene segments comprise Vκ2-24, Vκ3-20, Vκ1-17,    Vκ1-16, Vκ3-15, Vκ1-13, Vκ1-12, Vκ3-11, Vκ1-9, Vκ1-8, Vκ1-6, Vκ1-5,    Vκ5-2, Vκ4-1, Jκ1, Jκ2, Jκ3, Jκ4 and Jκ5. Optionally wherein the    gene segments are the alleles of Table 12.-   90. The mouse of any one of aspects 70 to 89, wherein the human VH,    DH and JH gene segments comprise human V_(H) gene segments V_(H)2-5,    7-4-1, 4-4, 1-3, 1-2, 6-1, and all the human D and J_(H) gene    segments D1-1, 2-2, 3-3, 4-4, 5-5, 6-6, 1-7, 2-8, 3-9, 5-12, 6-13,    2-15, 3-16, 4-17, 6-19, 1-20, 2-21, 3-22, 6-25, 1-26 and 7-27; and    J1, J2, J3, J4, J5 and J6. For example, the human VH, DH and JH gene    segments comprise human V_(H) gene segments V_(H)2-5, 7-4-1, 4-4,    1-3, 1-2, 6-1, and all the human D and J_(H) gene segments D1-1,    2-2, 3-3, 4-4, 5-5, 6-6, 1-7, 2-8, 3-9, 3-10, 4-11, 5-12, 6-13,    1-14, 2-15, 3-16, 4-17, 5-18, 6-19, 1-20, 2-21, 3-22, 4-23, 5-24,    6-25, 1-26 and 7-27; and J1, J2, J3, J4, J5 and J6. Optionally    wherein the gene segments are the alleles of Table 7.-   91. Use of the mouse of any one of aspects 70 to 90 for expressing    immunoglobulin heavy chains comprising human variable regions,    wherein the heavy chains expressed by the mouse are essentially    exclusively said heavy chains comprising human variable regions; and    said heavy chains comprising human variable regions are expressed as    part of serum IgG1, IgG2b and IgM (and optionally IgG2a) antibodies    in the mouse. The use is non-therapeutic, non-diagnostic and    non-surgical use.

In one embodiment, the use comprises immunising the mouse with anantigen (eg, a human antigen) and isolating an IgG1 antibody thatspecifically binds the antigen.

In one embodiment, the use comprises immunising the mouse with anantigen (eg, a human antigen) and isolating an IgG2a antibody thatspecifically binds the antigen.

In one embodiment, the use comprises immunising the mouse with anantigen (eg, a human antigen) and isolating an IgG2b antibody thatspecifically binds the antigen. Optionally, the use comprises making aderivative of the isolated antibody. Examples of derivative antibodies(according to any aspect herein) are antibodies that have one or moremutations compared to the isolated antibody (eg, to improveantigen-binding affinity and/or to enhance or inactivate Fc function)Such mutants specifically bind the antigen.

-   92. Use of the mouse of any one of aspects 70 to 90 for expressing    immunoglobulin heavy chains comprising human variable regions,    wherein the heavy chains expressed by the mouse are essentially    exclusively said heavy chains comprising human variable regions and    are expressed in a mouse that produces a normal proportion or    percentage of mature splenic B-cells. The use is non-therapeutic,    non-diagnostic and non-surgical use.

In one embodiment, the use further comprises isolating splenic tissue(eg, the spleen) from the mouse; optionally followed by isolating atleast one antigen-specific B-cell from the tissue, wherein the B-cell(s)expresses an antibody that specifically binds a predetermined antigen.In one example, the use comprises immunising the mouse with the antigenprior to isolating the splenic tissue. In an example, the use comprisesisolating an antibody produced by the B-cell (or by a hybridoma producedby fusion of the B-cell with a myeloma cell). Optionally, the usecomprises making a derivative of the isolated antibody. Examples ofderivative antibodies (according to any aspect herein) are antibodiesthat have one or more mutations compared to the isolated antibody (eg,to improve antigen-binding affinity and/or to enhance or inactivate Fcfunction) Such mutants specifically bind the antigen.

-   93. Use of the mouse of any one of aspects 70 to 90 for expressing    immunoglobulin heavy chains comprising human variable regions,    wherein the heavy chains expressed by the mouse are essentially    exclusively said heavy chains comprising human variable regions and    are expressed in a mouse that produces a normal proportion or    percentage of bone marrow B-cell progenitor cells. The use is    non-therapeutic, non-diagnostic and non-surgical use.-   94. Use of the mouse of any one of aspects 70 to 90 for the purpose    stated in one or more of aspects 70, 71, 73, 75 and 78.

The expression (eg, percentage expression or expression proportion orlevel) of Ig can be determined at the level of antibody chain mRNAtranscripts in B-cells (eg, peripheral blood lymphocytes). Alternativelyor additionally, the percentage expression is determined at the level ofantibody in serum or blood of the vertebrates. Additionally oralternatively, the expression can be determined by FACS analysis of Bcells.

In these aspects, “heavy chains comprising human variable regions” meansvariable regions derived from the recombination of human VH, D and JHgene segments.

“Essentially exclusively” the expressed heavy chains comprise humanvariable regions, ie, there is only a relatively very low or even noendogenous mouse heavy chain variable region expression. For example, atleast 90, 95, 96, 97, 98, 99 or 99.5% or 100% of the heavy chains areheavy chains comprising human variable regions. In one embodiment, atleast 90% of the heavy chains are heavy chains comprising human variableregions. The percentage expression can be determined at the level ofheavy chain mRNA transcripts in B-cells (eg, peripheral bloodlymphocytes). Alternatively or additionally, the percentage expressionis determined at the level of heavy chains or antibodies in serum orblood of the mice. Additionally or alternatively, the expression can bedetermined by FACS analysis of B-cells.

The mouse can comprise any endogenous heavy chain locus in which humanV, D and J gene segments are present, as described herein. In oneexample, the mouse genome comprises a mouse heavy chain locus in whichat least human V_(H) gene segments V_(H)2-5, 7-4-1, 4-4, 1-3, 1-2, 6-1,and all the human D and J_(H) gene segments D1-1, 2-2, 3-3, 4-4, 5-5,6-6, 1-7, 2-8, 3-9, 5-12, 6-13, 2-15, 3-16, 4-17, 6-19, 1-20, 2-21,3-22, 6-25, 1-26 and 7-27; and J1, J2, J3, J4, J5 and J6 are upstream ofthe mouse constant region.

The vertebrate in these aspects is, for example naïve (ie, not immunisedwith a predetermined antigen, as the term is understood in the art; forexample, such a vertebrate that has been kept in a relatively sterileenvironment as provided by an animal house used for R&D). In anotherexample, the vertebrate has been immunised with a predetermined antigen,eg, an antigen bearing a human epitope.

In one embodiment, the heavy chains, together with light chainsexpressed in the mice of the invention, form antibodies (Ig). The lightchains can be expressed from any transgenic light chain locus as hereindescribed. For example the genome of the mouse comprises a heavy chainlocus in which is a chimaeric immunoglobulin heavy chain locuscomprising one or more human V gene segments, one or more human D genesegments and one or more human J gene segments upstream of a mu constantregion of said non-human species; endogenous heavy chain expression hasbeen substantially inactivated; and the heavy chain locus comprises anEp enhancer of said non-human vertebrate species.

In one embodiment of any aspect, endogenous light chain (eg, kappaand/or lambda) expression is substantially inactive or inactivated, forexample using method as described herein. In this case, less than 10, 5,4, 3, 2, 1 or 0.5% of such endogenous lambda light chains are expressedor expressible. Additionally or alternatively, less than 10, 5, 4, 3, 2,1 or 0.5% of such endogenous kappa light chains are expressed orexpressible. In one example, there is complete inactivation ofendogenous kappa and/or lambda expression so no such light chains areexpressed or expressible.

In one embodiment, the genome of the mouse comprises human kappa genesegments (optionally the alleles of Table 12)

-   -   (i) Vκ1-5, Vκ1-6, Vκ1-8 and Vκ1-9 (and optionally Vκ5-2 and        Vκ4-1); or    -   (ii) Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ3-11, Vκ1-12, Vκ3-15, Vκ1-16,        Vκ1-17, Vκ3-20 (and optionally Vκ 2-24 and/or Vκ1-13); or    -   (iii) Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ3-11, Vκ1-12, Vκ3-15,        Vκ1-16, Vκ1-17, Vκ3-20, Vκ 2-24, Vκ1-27, Vκ2-28, Vκ2-30 and        Vκ1-33 (and optionally Vκ 2-29 and/or Vκ2-40 and/or Vκ1-39);    -   and optionally    -   (iv) Jκ1, Jκ2, Jκ3, Jκ4 and Jκ5.

In one embodiment, the genome also comprises (i) at least human V_(H)gene segments V_(H)2-5, 7-4-1, 4-4, 1-3, 1-2, 6-1, and all the human Dand J_(H) gene segments D1-1, 2-2, 3-3, 4-4, 5-5, 6-6, 1-7, 2-8, 3-9,5-12, 6-13, 2-15, 3-16, 4-17, 6-19, 1-20, 2-21, 3-22, 6-25, 1-26 and7-27; and J1, J2, J3, J4, J5 and J6 (optionally the alleles of Table 7)and (ii) at least human gene segments Vκ2-24, Vκ3-20, Vκ1-17, Vκ1-16,Vκ3-15, Vκ1-13, Vκ1-12, Vκ3-11, Vκ1-9, Vκ1-8, Vκ1-6, Vκ1-5, Vκ5-2,Vκ4-1, Jκ1, Jκ2, Jκ3, Jκ4 and Jκ5 (optionally the alleles of Table 12).As demonstrated in Example 16, such mice are fully functional in theaspect of rearrangement, BCR signalling and B cell maturation. Greaterthan 90% of the antibodies expressed by the mice comprised human heavychain variable regions and human kappa light chain variable regions.These mice are, therefore, very useful for the selection of antibodieshaving human variable regions that specifically bind human antigenfollowing immunisation of the mice with such antigen. Followingisolation of such an antibody, the skilled person can replace the mouseconstant regions with human constant regions using conventionaltechniques to arrive at totally human antibodies which are useful asdrug candidates for administration to humans (optionally followingmutation or adaptation to produce a further derivative, eg, with Fcenhancement or inactivation or following conjugation to a toxic payloador reporter or label or other active moiety).

In one embodiment, the genome also comprises a human iEκ and/or human3′Eκ downstream of the human J gene segments in the locus.

The Invention Also Includes the Following Clauses:

Clause 1. A mouse that expresses immunoglobulin heavy chains containinghuman variable regions,

-   -   wherein the mouse comprises a genome that includes an        immunoglobulin heavy chain locus comprising human VH, DH, and JH        gene segments positioned upstream to a mouse constant region;    -   wherein the mouse expresses immunoglobulin heavy chains,        characterized in that at least 90% of the immunoglobulin heavy        chains expressed by the mouse comprise a human variable region;        and    -   wherein the mouse expresses serum IgG1, IgG2b, and IgM        antibodies comprising said heavy chains containing a human        variable region.

Clause 2. A mouse that expresses immunoglobulin heavy chains containinghuman variable regions,

-   -   wherein the mouse comprises a genome that includes an        immunoglobulin heavy chain locus comprising human VH, DH, and JH        gene segments which are positioned upstream to a mouse constant        region;    -   wherein the mouse expresses immunoglobulin heavy chains,        characterized in that at least 90% of the immunoglobulin heavy        chains expressed by the mouse comprise a human variable region;        and    -   wherein the mouse produces a normal proportion of mature splenic        B-cells;    -   wherein said normal proportion is a proportion of mature splenic        B-cells produced by a mouse that expresses immunoglobulin heavy        chains containing mouse variable regions and does not express        immunoglobulin heavy chains containing human variable regions.

Clause 3. A mouse that expresses immunoglobulin heavy chains containinghuman variable regions,

-   -   wherein the mouse comprises a genome that includes an        immunoglobulin heavy chain locus comprising human VH, DH, and JH        gene segments which are positioned upstream to a mouse constant        region;    -   wherein the mouse expresses immunoglobulin heavy chains,        characterized in that it at least 90% of the immunoglobulin        heavy chains expressed by the mouse comprise a human variable        region; and    -   wherein the mouse produces a normal proportion of bone marrow        B-cell progenitor cells;    -   wherein the normal proportion is a proportion of bone marrow        B-cell progenitor cells produced by a mouse that expresses        immunoglobulin heavy chains containing mouse variable regions        and does not expresses immunoglobulin heavy chains containing        human variable regions.

Clause 4. The mouse of any of the preceding clauses, wherein the mouseexpresses a normal proportion of IgG1, IgG2b, and IgM in a sample ofserum obtained from the mouse;

-   -   wherein the normal proportion is as produced by a mouse that        expresses immunoglobulin heavy chains containing mouse variable        regions and does not expresses immunoglobulin heavy chains        containing human variable regions.

Clause 5. The mouse of any of the preceding clauses, wherein the mouseconstant region is C-mu, C-delta, and/or C-gamma.

Clause 6. The mouse of clause 5, wherein the mouse constant region is atleast C-mu, C-delta and C-gamma.

Clause 7. The mouse of any of the preceding clauses, wherein the mouseconstant region is an endogenous mouse C-region.

Clause 8. The mouse of any of the preceding clauses, wherein the mouseexpresses a human C-gamma region.

Clause 9. The mouse of any of the preceding clauses, wherein the mouseis a naïve mouse.

Clause 10. The mouse of clause 1, wherein the mouse expresses serumIgG2a comprising said heavy chains containing a human variable region.

Clause 11. The mouse of any of the preceding clauses, wherein the mouseexpresses Ig subtypes in a relative proportion of

-   -   (i) serum IgG1 at a concentration of about 25-350 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-200 μg/ml;    -   (iii) serum IgG2b at a concentration of about 30-800 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-300 μg/ml;    -   Or    -   (i) serum IgG1 at a concentration of about 10-600 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-500 μg/ml;    -   (iii) serum IgG2b at a concentration of about 20-700 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-700 μg/ml;    -   as determined by immunoglobulin capture on a plate followed by        incubation with an anti-mouse isotype-specific antibodies each        comprising a label and quantification of each immunoglobulin        based on the level of each label.

Clause 12. The mouse of any of the preceding clauses, wherein the mouseexpresses Ig subtypes in a relative proportion of

-   -   (i) total serum IgG and IgM at a concentration of about 200-2500        μg/ml;    -   and    -   (ii) serum IgM at a concentration of about 100-800 μg/ml;    -   as determined by immunoglobulin capture on a plate followed by        incubation with an anti-mouse isotype-specific antibodies each        comprising a label and quantification of each immunoglobulin        based on the level of each label.

Clause 13. The mouse of any of the preceding clauses, wherein the mouseexpresses said immunoglobulin heavy chains from splenic B-cells andwherein the mouse produces a normal proportion of mature splenic B-cellsin total spleen cells comprising mature B-cells, and splenic T1 and T2cells.

Clause 14. The mouse of any one of clauses 1-3, wherein, at least 95,96, 97, 98, 99, or 99.5% of the immunoglobulin heavy chains expressed bythe mouse are immunoglobulin heavy chains comprising human variableregions.

Clause 15. The mouse of any of the preceding clauses, wherein a mouseimmunoglobulin heavy chain enhancer is positioned in said mouse heavychain immunoglobulin locus between the human VH, DH, and JH genesegments and the mouse constant region.

Clause 16. The mouse of any of the preceding clauses, wherein a mouseS-mu switch is positioned in said mouse heavy chain immunoglobulin locusbetween the human VH, DH, and JH gene segments and the mouse constantregion.

Clause 17. The mouse of any of the preceding clauses, wherein endogenousmouse immunoglobulin heavy chain V, D and J gene segments are positionedin said mouse heavy chain immunoglobulin locus upstream to the human VH,DH, and JH gene segments.

Clause 18. The mouse of clause 17, wherein the mouse immunoglobulinheavy chain V, D and J gene segments are present in said mouse heavychain immunoglobulin locus with endogenous inter-gene segment sequences.

Clause 19. The mouse of clause 17 or 18, wherein the mouseimmunoglobulin heavy chain V, D and J gene segments are positioned insaid mouse heavy chain immunoglobulin locus in an orientation that isinverted relative to its natural endogenous orientation.

Clause 20. The mouse of any of the preceding clauses, wherein the mouseexpresses light chains containing human kappa variable regions.

Clause 21. The mouse of clause 20, wherein the mouse expressesimmunoglobulin light chains derived from recombination of Vκ with humanJκ.

Clause 22. The mouse of any of the preceding clauses, wherein the mouseexpresses light chains containing human lambda variable regions.

Clause 23. The mouse of clause 22, wherein the mouse expressesimmunoglobulin light chains derived from recombination of Vλ with humanJλ.

Clause 24. The mouse of clause 21, comprising a genome that includeshuman Vκ and Jκ gene segments positioned in said mouse heavy chainimmunoglobulin locus upstream to a mouse CL.

Clause 25. The mouse of clause 24, wherein the mouse CL is an endogenousCκ.

Clause 26. The mouse of clauses 24 or 25, wherein the human Vκ and Jκgene segments comprise Vκ2-24, Vκ3-20, Vκ1-17, Vκ1-16, Vκ3-15, Vκ1-13,Vκ1-12, Vκ3-11, Vκ1-9, Vκ1-8, Vκ1-6, Vκ1-5, Vκ5-2, Vκ4-1, Jκ1, Jκ2, Jκ3,Jκ4 and Jκ5.

Clause 27. The mouse of any the preceding clauses, wherein the human VH,DH and JH gene segments contain

-   -   human VH gene segments: VH2-5, 7-4-1, 4-4, 1-3, 1-2, 6-1;    -   human DH gene segments: D1-1, 2-2, 3-3, 4-4, 5-5, 6-6, 1-7, 2-8,        3-9, 5-12, 6-13, 2-15, 3-16, 4-17, 6-19, 1-20, 2-21, 3-22, 6-25,        1-26 and 7-27; and    -   human JH gene segments: J1, J2, J3, J4, J5 and J6.

Clause 28. A method for obtaining one or more immunoglobulin heavychains containing human variable regions, comprising providing the mouseof any of the preceding clauses and

isolating one or more immunoglobulin heavy chains.

Clause 29. The method of clause 28, wherein each immunoglobulin heavychain is included in an antibody.

Clause 30. The method of clause 29, wherein said heavy chain and/or saidantibody containing said heavy chain is modified after said isolating.

Clause 31. The method of clause 28, wherein a step of immunizing themouse with an antigen is performed before the step of isolating theimmunoglobulin heavy chains.

Clause 31a. The method of clause 30, wherein the antigen is a humanantigen.

Clause 32. The method of clause 30, 31, or 31a, wherein theimmunoglobulin heavy chains are included in an IgG1 antibody, antibodyfragment, or antibody derivative that specifically binds the antigen.

Clause 33. The method of clause 30, 31, or 31a, wherein theimmunoglobulin heavy chains are included in an IgG2a antibody, antibodyfragment, or antibody derivative that specifically binds the antigen.

Clause 34. The method of clause 30, 31, or 31a, wherein theimmunoglobulin heavy chains are included in an IgG2b antibody, antibodyfragment, or antibody derivative that specifically binds the antigen.

Clause 35. The method of clause 30, 31, or 31a, wherein theimmunoglobulin heavy chains are included in an IgM antibody, antibodyfragment, or antibody derivative that specifically binds the antigen.

Clause 36. An antibody or immunoglobulin heavy chain isolated in themethod of any one of clauses 28 to 35, or a antigen-binding fragment orderivative of the antibody or heavy chain.

Clause 37. A pharmaceutical composition comprising the antibody,antibody fragment, or antibody derivative of clause 36 and apharmaceutically acceptable carrier, excipient, or diluent.

Clause 38. A method for isolating splenic tissue comprising providingthe mouse of 1 to 27,

-   -   collecting a spleen or portion thereof from the mouse, and    -   obtaining tissue from the spleen or portion.

Clause 39. The method of clause 38, further comprising isolating atleast one antigen-specific B-cell from the splenic tissue, wherein theB-cell expresses a heavy chain containing a human variable region.

Clause 40. The method of clause 38 or 39, wherein a step of immunizingthe mouse with an antigen is performed before the step of collecting aspleen from the mouse.

Clause 41. The method of clause 40, wherein the antigen is a humanantigen.

Clause 42. The method of clause 40 or 41 wherein the at least oneantigen-specific B-cell produces an IgG1, IgG2a, IgG2b or IgM antibodycomprising said heavy chain, wherein the antibody specifically binds theantigen.

Clause 43. The method of clauses 38 to 42, wherein the at least oneantigen-specific B-cell that produces said heavy chain is fused with animmortal myeloma cell to produce a hybridoma cell.

Clause 44. The method of clauses 38 to 43, further comprising a step ofisolating an immunoglobulin heavy chain from the B-cell or the hybridomacell.

Clause 45. An antibody or immunoglobulin heavy chain isolated in themethod of clause 44, or a antigen-binding fragment or derivative of theantibody or heavy chain.

Clause 46. A pharmaceutical composition comprising the antibody,antibody fragment, or antibody derivative of clause 45 and apharmaceutically acceptable carrier, excipient, or diluent.

Clause 47. A method for obtaining a humanised antibody, comprising

-   -   selecting a mouse that expresses immunoglobulin heavy chains        containing human variable regions,    -   wherein the mouse comprises a genome that includes an        immunoglobulin heavy chain locus comprising human VH, DH, and JH        gene segments positioned upstream to a mouse constant region,    -   wherein the mouse expresses immunoglobulin heavy chains,        characterized in that at least 90% of the immunoglobulin heavy        chains expressed by the mouse are immunoglobulin heavy chains        containing a human variable region,    -   wherein the mouse expresses serum IgG1, IgG2b, and IgM        antibodies comprising said heavy chains containing a human        variable region,    -   wherein the mouse produces a normal proportion of mature splenic        B-cells,    -   wherein the mouse produces a normal proportion of bone marrow        B-cell progenitor cells, and    -   wherein the mouse expresses a normal proportion of IgG1, IgG2a,        IgG2b, and IgM in a sample of serum obtained from the mouse, and    -   wherein each said normal proportion is a proportion produced by        a mouse that expresses immunoglobulin heavy chains containing        mouse variable regions and does not expresses immunoglobulin        heavy chains containing human variable regions;    -   collecting serum from said mouse; and    -   obtaining a pool of humanised antibodies comprising IgG1, IgG2b,        and IgM antibodies from the serum.

Clause 48. The method of clause 47, comprising a step of immunizing themouse with an antigen before the step of collecting serum from saidmouse.

Clause 49. The method of clause 48, further comprising steps of

-   -   contacting said pool of humanised antibodies with said antigen;    -   binding said antigen with a humanised antibody in said pool of        humanised antibodies; and    -   isolating the humanised antibody that binds to said antigen.

Clause 50. The method of clause 49, further comprising steps of

-   -   contacting the humanised antibody that binds to said antigen        with an isotype-specific antibody, wherein the isotype-specific        antibody recognizes IgG1, IgG2a, IgG2b, or IgM; and    -   isolating the humanised antibody that binds to said        isotype-specific antibody.

Clause 51. The method of clause 48, further comprising the steps of

-   -   collecting the spleen or tissue thereof from said mouse,    -   isolating B-cells from splenic tissue,    -   fusing said B-cells with immortal myeloma cells to produce        hybridoma cells    -   expressing a pool of humanised antibodies comprising IgG        antibodies from the    -   serum, wherein the pool of antibodies is used in the method of        clause 48.

Clause 52. The method of any of clauses 47-51, wherein said selectedmouse comprises mouse immunoglobulin heavy chain V, D and J genesegments which are positioned in said mouse heavy chain immunoglobulinlocus in an orientation that is inverted relative to its naturalendogenous orientation.

Clause 53. The method of any of clauses 47-52 wherein the mouseexpresses Ig subtypes in a relative proportion of

-   -   (i) serum IgG1 at a concentration of about 25-350 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-200 μg/ml;    -   (iii) serum IgG2b at a concentration of about 30-800 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-300 μg/ml;    -   Or    -   (i) serum IgG1 at a concentration of about 10-600 μg/ml;    -   (ii) serum IgG2a at a concentration of about 0-500 μg/ml;    -   (iii) serum IgG2b at a concentration of about 20-700 μg/ml; and    -   (iv) serum IgM at a concentration of about 50-700 μg/ml;        as determined by immunoglobulin capture on a plate followed by        incubation with an anti-mouse isotype-specific antibodies each        comprising a label and quantification of each immunoglobulin        based on the level of each label.

Clause 54. The method of any one of clauses 47 to 53, wherein, at least95, 96, 97, 98, 99, or 99.5% of the immunoglobulin heavy chainsexpressed by the mouse are immunoglobulin heavy chains comprising humanvariable regions.

Clause 55. The method of any clauses 47-54, wherein a mouseimmunoglobulin heavy chain enhancer is positioned in said mouse heavychain immunoglobulin locus between the human VH, DH, and JH genesegments and the mouse constant region.

Clause 56. The method of any of clauses 47-55, wherein a mouse S-muswitch is positioned in said mouse heavy chain immunoglobulin locusbetween the human VH, DH, and JH gene segments and the mouse constantregion.

Clause 57. The method of any of clauses 47-56, wherein endogenous mouseimmunoglobulin heavy chain V, D and J gene segments are positioned insaid mouse heavy chain immunoglobulin locus upstream to the human VH,DH, and JH gene segments.

Clause 58. The method of clause 57, wherein the mouse immunoglobulinheavy chain V, D and J gene segments are present in said mouse heavychain immunoglobulin locus with endogenous inter-gene segment sequences.

Clause 59. The method of clause 57 or 58, wherein the mouseimmunoglobulin heavy chain V, D and J gene segments are positioned insaid mouse heavy chain immunoglobulin locus in an orientation that isinverted relative to its natural endogenous orientation.

Clause 60. The method of any of clauses 47-59, wherein the mouseexpresses light chains containing human kappa variable regions.

Clause 61. The method of clause 60, wherein the mouse expressesimmunoglobulin light chains containing human Jκ.

Clause 62. The method of any of clauses 47-51, wherein the mouseexpresses light chains containing human lambda variable regions.

Clause 63. The method of clause 62, wherein the mouse expressesimmunoglobulin light chains containing human Jλ.

Clause 64. The method of clause 61, comprising a genome that includeshuman Vκ and Jκ gene segments positioned in said mouse heavy chainimmunoglobulin locus upstream to a mouse CL.

Clause 65. The mouse of clause 64, wherein the mouse CL is an endogenousCκ.

Clause 66. The mouse of clauses 64 or 65, wherein the human Vκ and Jκgene segments comprise Vκ2-24, Vκ3-20, Vκ1-17, Vκ1-16, Vκ3-15, Vκ1-13,Vκ1-12, Vκ3-11, Vκ1-9, Vκ1-8, Vκ1-6, Vκ1-5, Vκ5-2, Vκ4-1, Jκ1, Jκ2, Jκ3,Jκ4 and Jκ5.

Clause 67. The method of any of clauses 47-51, wherein the human VH, DHand JH gene segments contain

-   -   human VH gene segments: VH2-5, 7-4-1, 4-4, 1-3, 1-2, 6-1;    -   human DH gene segments: D1-1, 2-2, 3-3, 4-4, 5-5, 6-6, 1-7, 2-8,        3-9, 5-12, 6-13, 2-15, 3-16, 4-17, 6-19, 1-20, 2-21, 3-22, 6-25,        1-26 and 7-27; and    -   human JH gene segments: J1, J2, J3, J4, J5 and J6.

Non-Human Vertebrates Expressing Kappa & Lambda Variable Regions (i) Kand L Chains Produced in Human-Like Ratios

This aspect of the invention is useful for producing light chains thatare not skewed to non-human-like ratios. For example, in mice kappa-typelight chains predominate by far over lambda-type light chains (typicallyof the order of 95% kappa light chains: 5% lambda light chains in awild-type mouse). Humans, on the other hand, typically display around60% kappa:around 40% lambda. Thus, lambda expression is much higher thanfound in a mouse. It would be desirable to provide a non-humanvertebrate, such as a mouse or a rat, in which a higher proportion oflambda-type light chains can be expressed. This is useful when thevertebrate expresses light chains bearing human lambda variable regionsand other light chains bearing human kappa variable regions. To thisend, the inventors have demonstrated for the first time such avertebrate that expresses elevated lambda light chains, and thus theinvention provides:—

A non-human vertebrate (eg, a mouse or rat) whose genome comprises an Iggene segment repertoire produced by targeted insertion of human Ig genesegments into one or more endogenous Ig loci, the genome comprisinghuman Vλ and Jλ gene segments provided by insertion into an endogenouslight chain locus of the vertebrate upstream of a constant region, thegenome comprising human Vκ and Jκ gene segments provided by insertioninto an endogenous light chain locus of the vertebrate upstream of aconstant region, wherein the vertebrate expresses immunoglobulin lightchains comprising kappa light chain variable regions and immunoglobulinlight chains comprising lambda light chain variable regions, whereinmore than 20% of the light chains expressed by the vertebrate compriselambda variable regions (eg, as determined by FACS of splenic B cells).

The remaining light chains express kappa variable regions.

WO03047336 teaches the desirability of producing human-like kappa:lambdaratios, but this does not provide an enabled or plausible disclosure ofhow to achieve this.

(ii) K and L Chains Produced with Normal B-Cell Compartments

The inventors have successfully generated non-human vertebratescontaining targeted insertion of human V and J lambda gene segments toenable expression of light chains comprising human lambda variableregions by normal (ie, comparable to wild-type vertebrate) B-cellcompartments. Thus, the inventors have provided such vertebrates thatcan usefully produce such light chains with good repertoires and morereliably than prior art transgenic non-human vertebrates that displaycomprised B-cell compartments of reduced size and maturity, and indeedwhich may not even produce light chains having human lambda variableregions. Thus, the invention provides:—

A non-human vertebrate (eg, a mouse or rat) whose genome comprises an Iggene segment repertoire produced by targeted insertion of human Ig genesegments into one or more endogenous Ig loci, the genome comprisinghuman Vλ and Jλ gene segments provided by insertion into an endogenouslight chain locus of the vertebrate upstream of a constant region, thegenome comprising human Vκ and Jκ gene segments provided by insertioninto an endogenous light chain locus of the vertebrate upstream of aconstant region, wherein the vertebrate expresses immunoglobulin lightchains comprising kappa light chain variable regions and immunoglobulinlight chains comprising lambda light chain variable regions, and whereinthe vertebrate produces a normal proportion or percentage of maturesplenic B-cells (eg, as determined by FACS of splenic B cells).

With regard to non-human vertebrates (i) and (ii), the followingembodiments are contemplated (unless specified, each embodiment appliesto (i) or (ii)):—

In an embodiment, the human Vλ and Jλ insertion comprises at least thefunctional human V and J gene segments comprised by a human lambda chainIg locus from Vλ3-27 to Cλ7.

In an embodiment, the human Vλ and Jλ insertion comprises at least humanV gene segments Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18,Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 and Vλ3-1,optionally the alleles of Table 18.

In an embodiment, the human Vλ and Jλ insertion comprises one, more orall of human J gene segments Jλ1, Jλ2, Jλ3, Jλ6 and Jλ7.

In an embodiment, the human Vλ and Jλ insertion comprises an insertionof a human Jλ-Cλ cluster, wherein the cluster comprises the J and C genesegments from Jλ1 to Cλ7.

In an embodiment, the human Vλ and Jλ insertion comprises an insertionof a human EA enhancer. For example, the EA enhancer is provided ingermline configuration with respect to a human Jλ7 that is alsocomprised by the insertion. For example, the EA enhancer is provided ingermline configuration with respect to a human Jλ-Cλ cluster that isalso comprised by the insertion, wherein the cluster comprises Jλ1 toCλ7 in human germline configuration. In a human germline configurationthe EA enhancer is 3′ of the Jλ-Cλ cluster.

In an embodiment or vertebrate (i) or (ii), the human Vλ and Jλinsertion is provided by an insertion of a sequence corresponding tocoordinates 22886217 to 23327884 of human chromosome 22.

In an embodiment or vertebrate (ii), the human Vλ and Jλ insertion isprovided by an insertion of a sequence corresponding to coordinates23064876 to 23327884 of human chromosome 22.

In an embodiment, the human Vκ and Jκ insertion comprises at least thefunctional human V and J gene segments comprised by a human kappa chainIg locus from Vκ1-33 to Jκ5.

In an embodiment, the human Vκ and Jκ insertion comprises at least humanV gene segments Vκ1-33, Vκ2-30, Vκ2-29, Vκ2-28, Vκ1-27, Vκ2-24, Vκ3-20,Vκ1-17, Vκ1-16, Vκ3-15, Vκ1-13, Vκ1-12, Vκ3-11, Vκ1-9, Vκ1-8, Vκ1-6,Vκ1-5, Vκ5-2 and Vκ4-1, optionally the alleles of Table 12.

In an embodiment, the human Vκ and Jκ insertion comprises one, more orall of human J gene segments Jκ1, Jκ2, Jκ3, Jκ4 and Jκ5, optionally thealleles of Table 12.

In an embodiment, more than 30, 35, 40, 45 or 50% of the light chainsexpressed by the vertebrate comprise lambda variable regions.

In an embodiment, from 20 to 40, 45 or 50% of the light chains expressedby the vertebrate comprise lambda variable regions. In an embodiment,from 30 to 40, 45 or 50% of the light chains expressed by the vertebratecomprise lambda variable regions.

In an embodiment, said kappa light chain variable regions are humankappa light chain variable regions.

In an embodiment, the human Vκ and Jκ gene segments are in an endogenouskappa light chain locus of the vertebrate upstream of a kappa constantregion.

In an embodiment, the human Vλ and Jλ gene segments are in an endogenouskappa light chain locus of the vertebrate.

In an embodiment, the human Vλ and Jλ gene segments are in an endogenouslambda light chain locus of the vertebrate.

In an embodiment, the vertebrate expresses light chains comprising humankappa variable regions and expresses light chains comprising humanlambda variable regions. In an example, endogenous (non-humanvertebrate) kappa chain expression is substantially inactive or isinactive and/or endogenous (non-human vertebrate) lambda chainexpression is substantially inactive or is inactive. Where thevertebrate is a mouse, mouse lambda chain expression is typically verylow (around 5% or less) and in this case it may not be necessary toengineer the mouse genome to further inactivate endogenous lambda chainexpression. Thus, where the vertebrate is s mouse, endogenous kappachain expression is substantially inactive or is inactive and mouselambda chain expression is 5% or less of all light chain expression.

In an embodiment, the vertebrate produces a normal proportion orpercentage of mature splenic B-cells. For example, this can bedetermined by FACS of splenic B cells isolated from the vertebrate.

In an embodiment, the vertebrate produces a normal ratio of T1, T2 andmature splenic B-cells. For example, this can be determined by FACS ofsplenic B cells isolated from the vertebrate.

In an embodiment, at least 40, 50, 60 or 70% of total splenic B-cellsproduced by the vertebrate are mature B-cells. For example, this can bedetermined by FACS of splenic B cells isolated from the vertebrate.

Further Statements of Invention

In one embodiment the invention relates to the following:

A non-human vertebrate (eg, a mouse or rat) or vertebrate cell whosegenome comprises human VH, D and JH gene segments upstream of a constantregion at a heavy chain locus and/or human VL and JL gene segmentsupstream of a constant region at a light chain locus, wherein the genesegments are operably linked to the constant region thereof so that thevertebrate or cell is capable of expressing immunoglobulin heavy and/orlight chains comprising human VH and VL domains respectively, whereinthe heavy chain locus comprises a human VH gene segment capable ofrecombining with a human D and JH gene segment to produce a VH domain,wherein the light chain locus comprises a human VL gene segment capableof recombining with a human JL gene segment to produce a VL domain, orwherein the cell can develop into a vertebrate that expresses said heavyand light chain variable domains.

A non-human vertebrate (eg, a mouse or rat) or vertebrate cell whosegenome comprises human VH, D and JH gene segments upstream of a constantregion at a heavy chain locus, wherein the gene segments are operablylinked to the constant region thereof so that the vertebrate or cell iscapable of expressing immunoglobulin heavy chains comprising human VHdomains, wherein the heavy chain locus comprises a human 01 allele VHgene segment capable of recombining with a human D and JH gene segmentto produce a VH domain, or wherein the cell can develop into avertebrate that expresses said heavy chain variable domain.

A non-human vertebrate (eg, a mouse or rat) or vertebrate cell whosegenome comprises VL and JL gene segments upstream of a constant regionat a light chain locus, wherein the gene segments are operably linked tothe constant region thereof so that the vertebrate or cell is capable ofexpressing immunoglobulin light chains comprising human VL domains,wherein the light chain locus comprises a human 01 allele VL genesegment capable of recombining with a human JL gene segment to produce aVL domain, or wherein the cell can develop into a vertebrate thatexpresses said light chain variable domain.

Optionally the vertebrate or cell has a genome comprising the heavychain locus and the light chain locus defined above and thereforecomprises a heavy chain locus comprising a human 01 allele VH genesegment capable of recombining with a human D and JH gene segment toproduce a VH domain and a light chain locus comprising a human 01 alleleVL gene segment capable of recombining with a human JL gene segment toproduce a VL domain.

In an alternative embodiment, the invention relates to a non-humanvertebrate or vertebrate cell (e.g. a mouse cell or rat cell) whosegenome comprises one or more human VH gene segments, one or more humanJH gene segments and one or more human D gene segments upstream of aconstant region at a heavy chain, wherein the gene segments are operablylinked to the constant region thereof so that the cell or vertebrate iscapable of producing an antibody heavy chain, or where the cell candevelop into a vertebrate that expresses an antibody heavy chain,wherein said one or more human VH gene segments of the heavy chain locuscomprise or consist of one, more or all human VH gene segments selectedfrom the group consisting of VH3-23*04, VH7-4-1*01, VH4-4*02, VH1-3*01,VH3-13*01, VH3-7*01, VH3-20*d01 and VH3-9*01.

In an embodiment of the invention, the VH gene segments are selectedfrom the group consisting of VH3-23*04, VH7-4-1*01, VH4-4*02, VH1-3*01,VH3-13*01, VH3-7*01 and VH3-20*d01.

For embodiments of the invention, which, for example, define human heavychain gene segments only, the light chain locus can comprise rearrangedor unrearranged VL and JL gene segments, e.g. a single rearranged VJ(such as a single rearranged Vκ1-39/J). Additionally or alternativelythe light chain locus can be randomly integrated. In another embodimentthe VL and JL segments are upstream of an endogenous constant light genesegment.

In a further alternative embodiment, the invention relates to anon-human vertebrate or vertebrate cell (e.g. a mouse cell or rat cell)whose genome comprises one or more human Jκ gene segments and one ormore human Vκ gene segments upstream of a constant region at a lightchain locus, wherein the gene segments are operably linked to theconstant region thereof so that the cell or vertebrate is capable ofproducing an antibody light chain, or where the cell can develop into avertebrate that expresses an antibody light chain, wherein said one ormore human Vκ gene segments comprise or consist of one, more or allhuman Vκ gene segments selected from the group consisting of Vκ4-1*01,Vκ2-28*01, Vκ1D-13*d01, Vκ1-12*01, Vκ1D-12*02, Vκ3-20*01, Vκ1-17*01,Vκ1D-39*01, Vκ3-11*01, Vκ1D-16*01 and Vκ1-9*d01.

In an alternative embodiment, the invention relates to a non-humanvertebrate (eg, a mouse or rat) or vertebrate cell whose genomecomprises human VH, D and JH gene segments upstream of a constant regionat a heavy chain locus, wherein the JH gene segments comprise JH1*01,JH2*01, JH3*02, JH4*02, JH5*02 and/or JH6*01 or JH6*02 and the genesegments are operably linked to the constant region thereof so that thevertebrate or cell is capable of expressing immunoglobulin heavy chainscomprising human VH domains, wherein the heavy chain locus comprises ahuman VH gene segment capable of recombining with a human D and one ofsaid JH gene segments to produce a VH domain, or wherein the cell candevelop into a vertebrate that expresses said heavy chain variabledomain.

In a preferred embodiment, the JH gene segments are JH1*01, JH2*01,JH3*02, JH4*02, JH5*02 and JH6*02. In another embodiment, the JH genesegments are JH1*01, JH2*01, JH3*02, JH4*02, JH5*02 and JH6*01.

In one embodiment, the non-human vertebrate further comprises one ormore of the VH gene segments and/or one or more of the D gene segmentsfrom Table 7. In a further embodiment, the non-human vertebrate furthercomprises the VH gene segments and D gene segments from Table 3.

A non-human vertebrate (eg, a mouse or rat) or vertebrate cell whosegenome comprises VL and JL gene segments upstream of a constant regionat a light chain locus, wherein the JL gene segments comprise Jκ1*01,Jκ2*01, Jκ3*01, Jκ4*01 and/or Jκ5*01 and the gene segments are operablylinked to the constant region thereof so that the vertebrate or cell iscapable of expressing immunoglobulin light chains comprising human VLdomains, wherein the light chain locus comprises a human VL gene segmentcapable of recombining with one of said human JL gene segments toproduce a VL domain, or wherein the cell can develop into a vertebratethat expresses said light chain variable domain.

In one embodiment, the non-human vertebrate further comprises one ormore or all Vκ gene segments from Table 12. In a further embodiment, thenon-human vertebrate further comprises the Vκ gene segments from Table10 or 11.

Optionally the vertebrate or cell has a genome comprising the heavychain locus and the light chain locus defined above and thereforecomprises a heavy chain locus comprising a human VH gene segment capableof recombining with a human D and one of said JH gene segments toproduce a VH domain and a light chain locus comprising a human VL genesegment capable of recombining with one of said human JL gene segmentsto produce a VL domain.

It is envisaged that in all embodiments of the invention, the genome ofthe cell or vertebrate in accordance with the invention may not comprisea second human allele of one, more or all of the human gene segment(s).

In all embodiments of the invention, the human JH gene segments, D genesegments and VH gene segments can be upstream of a constant region at anendogenous heavy chain locus and/or the human Jκ gene segments and Vκgene segments can be upstream of a constant region at an endogenouslight chain locus.

In one embodiment, the endogenous light chain locus is the endogenouskappa locus, and in another embodiment it is the endogenous lambdalocus.

Allele Combinations

In one embodiment, the allele of the gene segment is a d01 allele,optionally a d01 allele disclosed in Table 7, Table 12 or Table 18.

In one embodiment, vertebrate or cell has a genome further comprising a02, 03, 04, 05, 10, 12, 18 or d03 allele disclosed in Table 7, Table 12or Table 18.

The preferred alleles of the invention are set out in Tables 1 to 18 asfollows:

TABLE 1 IgH Alleles #1-S1 alleles Allele J_(H)6 02 J_(H)5 02 J_(H)4 02J_(H)3 02 J_(H)2 01 J_(H)1 01 D7-27 02 D1-26 01 D6-25 01 D5-24 01 D4-2301 D3-22 01 D2-21 02 D1-20 01 D6-19 01 D5-18 01 D4-17 01 D3-16 02 D2-1501 D1-14 01 D6-13 01 D5-12 01 D4-11 01 D3-10 01 D3-9  01 D2-2  02 D1-1 01 V_(H)6-1 01 V_(H)1-2 02 or 04 V_(H)1-3 01 V_(H)4-4 02 V_(H)7-4 01V_(H)2-5 01 or 10

In an alternative Table 1, the JH6 allele can be JH6*01.

TABLE 2 IgH Alleles #2-S2 alleles ID J_(H)6 02 J_(H)5 02 J_(H)4 02J_(H)3 02 J_(H)2 01 J_(H)1 01 D7-27 02 D1-26 01 D6-25 01 D5-24 01 D4-2301 D3-22 01 D2-21 02 D1-20 01 D6-19 01 D5-18 01 D4-17 01 D3-16 02 D2-1501 D1-14 01 D6-13 01 D5-12 01 D4-11 01 D3-10 01 D3-9  01 D2-2  02 D1-1 01 V_(H)6-1  01 V_(H)1-2  02 or 04 V_(H)1-3  01 V_(H)4-4  02 V_(H)7-4 01 V_(H)2-5  01 or 10 V_(H)3-7  01 V_(H)1-8  01 V_(H)3-9  01 V_(H)3-1101 V_(H)3-13 01

In an alternative Table 2, the JH6 allele can be JH6*01.

TABLE 3 IgH Alleles #3 - S3 alleles ID Allele J_(H)6 02 J_(H)5 02 J_(H)402 J_(H)3 02 J_(H)2 01 J_(H)1 01 D7-27 02 D1-26 01 D6-25 01 D5-24 01D4-23 01 D3-22 01 D2-21 02 D1-20 01 D6-19 01 D5-18 01 D4-17 01 D3-16 02D2-15 01 D1-14 01 D6-13 01 D5-12 01 D4-11 01 D3-10 01 D3-9 01 D2-2 02D1-1 01 V_(H)6-1 01 V_(H)1-2 02 or 04 V_(H)1-3 01 V_(H)4-4 02 V_(H)7-401 V_(H)2-5 01 or 10 V_(H)3-7 01 V_(H)1-8 01 V_(H)3-9 01 V_(H)3-11 01V_(H)3-13 01 V_(H)3-15 01 V_(H) 1-18 01 V_(H)3-20 01 or d01 V_(H)3-21 01or 03 V_(H)3-23 04 V_(H) 1-24 01 or d01 V_(H)2-26 01 or d01

In an alternative Table 3, the JH6 allele can be JH6*01.

TABLE 4 IgH Alleles #4 - S4 alleles ID Allele J_(H)6 02 J_(H)5 02 J_(H)402 J_(H)3 02 J_(H)2 01 J_(H)1 01 D7-27 02 D1-26 01 D6-25 01 D5-24 01D4-23 01 D3-22 01 D2-21 02 D1-20 01 D6-19 01 D5-18 01 D4-17 01 D3-16 02D2-15 01 D1-14 01 D6-13 01 D5-12 01 D4-11 01 D3-10 01 D3-9 01 D2-2 02D1-1 01 V_(H)6-1 01 V_(H)1-2 02 or 04 V_(H)1-3 01 V_(H)4-4 02 V_(H)7-401 V_(H)2-5 01 or 10 V_(H)3-7 01 V_(H)1-8 01 V_(H)3-9 01 V_(H)3-11 01V_(H)3-13 01 V_(H)3-15 01 V_(H)1-18 01 V_(H)3-20 01 or d01 V_(H)3-21 01or 03 V_(H)3-23 04 V_(H)1-24 01 or d01 V_(H)2-26 01 or d01 V_(H)4-28 05V_(H)3-30 18 V_(H)4-31 03 V_(H)3-33 01 V_(H)4-34 01 V_(H)4-39 01

In an alternative Table 4, the JH6 allele can be JH6*01.

TABLE 5 IgH Alleles #5 - S5 alleles ID Allele J_(H)6 02 J_(H)5 02 J_(H)402 J_(H)3 02 J_(H)2 01 J_(H)1 01 D7-27 02 D1-26 01 D6-25 01 D5-24 01D4-23 01 D3-22 01 D2-21 02 D1-20 01 D6-19 01 D5-18 01 D4-17 01 D3-16 02D2-15 01 D1-14 01 D6-13 01 D5-12 01 D4-11 01 D3-10 01 D3-9 01 D2-2 02D1-1 01 V_(H)6-1 01 V_(H)1-2 02 or 04 V_(H)1-3 01 V_(H)4-4 02 V_(H)7-401 V_(H)2-5 01 or 10 V_(H)3-7 01 V_(H)1-8 01 V_(H)3-9 01 V_(H)3-11 01V_(H)3-13 01 V_(H)3-15 01 V_(H)1-18 01 V_(H)3-20 01 or d01 V_(H)3-21 01or 03 V_(H)3-23 04 V_(H)1-24 01 or d01 V_(H)2-26 01 or d01 V_(H)4-28 05V_(H)3-30 18 V_(H)4-31 03 V_(H)3-33 01 V_(H)4-34 01 V_(H)4-39 01V_(H)3-43 01 V_(H)1-45 02 V_(H) 1-46 01 V_(H)3-48 01

In an alternative Table 5, the JH6 allele can be JH6*01.

TABLE 6 IgH Alleles #6 - S6 alleles ID Allele J_(H)6 02 J_(H)5 02 J_(H)402 J_(H)3 02 J_(H)2 01 J_(H)1 01 D7-27 02 D1-26 01 D6-25 01 D5-24 01D4-23 01 D3-22 01 D2-21 02 D1-20 01 D6-19 01 D5-18 01 D4-17 01 D3-16 02D2-15 01 D1-14 01 D6-13 01 D5-12 01 D4-11 01 D3-10 01 D3-9 01 D2-2 02D1-1 01 V_(H)6-1 01 V_(H)1-2 02 or 04 V_(H)1-3 01 V_(H)4-4 02 V_(H)7-401 V_(H)2-5 01 or 10 V_(H)3-7 01 V_(H)1-8 01 V_(H)3-9 01 V_(H)3-11 01V_(H)3-13 01 V_(H)3-15 01 V_(H)1-18 01 V_(H)3-20 01 or d01 V_(H)3-21 01or 03 V_(H)3-23 04 V_(H)1-24 01 or d01 V_(H)2-26 01 or d01 V_(H)4-28 05V_(H)3-30 18 V_(H)4-31 03 V_(H)3-33 01 V_(H)4-34 01 V_(H)4-39 01V_(H)3-43 01 V_(H)1-45 02 V_(H)1-46 01 V_(H)3-48 01 V_(H)3-49 05V_(H)5-51 01 V_(H)3-53 01 V_(H)1-58 01 V_(H)4-59 01 or 05 V_(H)4-61 01V_(H)3-64 02 V_(H)3-66 03 V_(H)1-69 12

In an alternative Table 6, the JH6 allele can be JH6*01.

TABLE 7 IgH Alleles #7 - S7 alleles (a complete repertoire of functionalIgH gene segments) ID Allele 1 J_(H)6 02 2 J_(H)5 02 3 J_(H)4 02 4J_(H)3 02 5 J_(H)2 01 6 J_(H)1 01 7 D7-27 02 8 D1-26 01 9 D6-25 01 10D5-24 01 11 D4-23 01 12 D3-22 01 13 D2-21 02 14 D1-20 01 15 D6-19 01 16D5-18 01 17 D4-17 01 18 D3-16 02 19 D2-15 01 20 D1-14 01 21 D6-13 01 22D5-12 01 23 D4-11 01 24 D3-10 01 25 D3-9 01 26 D2-2 02 27 D1-1 01 28V_(H)6-1 01 29 V_(H)1-2 02 or 04 30 V_(H)1-3 01 31 V_(H)4-4 02 32V_(H)7-4 01 33 V_(H)2-5 01 or 10 34 V_(H)3-7 01 35 V_(H)1-8 01 36V_(H)3-9 01 37 V_(H)3-11 01 38 V_(H)3-13 01 39 V_(H)3-15 1.1 01 40V_(H)1-18 01 41 V_(H)3-20 01 or d01 42 V_(H)3-21 01 or 03 43 V_(H)3-2304 44 V_(H)1-24 01 or d01 45 V_(H)2-26 01 or d01 46 V_(H)4-28 05 47V_(H)3-30 18 48 V_(H)4-31 03 49 V_(H)3-33 01 50 V_(H)4-34 01 51V_(H)4-39 01 52 V_(H)3-43 01 53 V_(H)1-45 02 54 V_(H)1-46 01 55V_(H)3-48 01 56 V_(H)3-49 05 57 V_(H)5-51 01 58 V_(H)3-53 01 59V_(H)1-58 01 60 V_(H)4-59 01 or 05 61 V_(H)4-61 01 62 V_(H)3-64 02 63V_(H)3-66 03 64 V_(H)1-69 12 65 V_(H)2-70 04 66 V_(H)3-72 01 67V_(H)3-73 02 68 V_(H)3-74 01

In an alternative Table 7, the JH6 allele can be JH6*01.

TABLE 8 Igκ Alleles #1 - K1 alleles ID Allele J_(K)5 01 J_(K)4 01 J_(K)301 J_(K)2 01 or 04 J_(K)1 01 V_(K)4-1 01 V_(K)5-2 01 or d01 V_(K)1-5 03V_(K)1-6 01 V_(K)1-8 01 V_(K)1-9 01 or d01

TABLE 9 Igκ Alleles #2-K2 alleles 1.2 ID Allele Jκ5 01 Jκ4 01 Jκ3 01 Jκ201 or 04 Jκ1 01 Vκ4-1 01 Vκ5-2 01 or d01 Vκ1-5 03 Vκ1-6 01 Vκ1-8 01Vκ1-9 01 or d01 Vκ3-11 01 Vκ1-12 01 Vκ1-13 01 Vκ3-15 01 Vκ1-16 02 Vκ1-1701 Vκ3-20 01 Vκ6-21 01 Vκ2-24 01

TABLE 10 Igκ Alleles #3-K3 alleles 1.3 ID Allele Jκ5 01 Jκ4 01 Jκ3 01Jκ2 01 or 04 Jκ1 01 Vκ4-1 01 Vκ5-2 01 or d01 Vκ1-5 03 Vκ1-6 01 Vκ1-8 01Vκ1-9 01 or d01 Vκ3-11 01 Vκ1-12 01 Vκ1-13 01 Vκ3-15 01 Vκ1-16 02 Vκ1-1701 Vκ3-20 01 Vκ6-21 01 Vκ2-24 01 Vκ1-27 01 Vκ2-28 01 Vκ2-29 01 Vκ2-30 01Vκ1-33 01 Vκ1D-39 01 Vκ2D-40 01

TABLE 11 Igκ Alleles #4-K4 alleles 1.4 ID Allele Jκ5 01 Jκ4 01 Jκ3 01Jκ2 01 or 04 Jκ1 01 Vκ4-1 01 Vκ5-2 01 or d01 Vκ1-5 03 Vκ1-6 01 Vκ1-8 01Vκ1-9 01 or d01 Vκ3-11 01 Vκ1-12 01 Vκ1-13 01 Vκ3-15 01 Vκ1-16 02 Vκ1-1701 Vκ3-20 01 Vκ6-21 01 Vκ2-24 01 Vκ1-27 01 Vκ2-28 01 Vκ2-29 01 Vκ2-30 01Vκ1-33 01 Vκ1D-39 01 Vκ2D-40 01 Vκ3D-7 01 Vκ1D-8 01 or d01 Vκ1D-43 01Vκ3D-11 01 or d01 Vκ1D-12 02 Vκ1D-13 d01 Vκ3D-15 d01 Vκ1D-16 01 Vκ1D-1701 Vκ3D-20 01

TABLE 12 Igκ Alleles #5-K5 alleles (a complete repertoire of functionalhuman kappa gene segments) 1.5 ID Allele Jκ5 01 Jκ4 01 Jκ3 01 Jκ2 01 or04 Jκ1 01 Vκ4-1 01 Vκ5-2 01 or d01 Vκ1-5 03 Vκ1-6 01 Vκ1-8 01 Vκ1-9 01or d01 Vκ3-11 01 Vκ1-12 01 Vκ1-13 01 Vκ3-15 01 Vκ1-16 02 Vκ1-17 01Vκ3-20 01 Vκ6-21 01 Vκ2-24 01 Vκ1-27 01 Vκ2-28 01 Vκ2-29 01 Vκ2-30 01Vκ1-33 01 Vκ1D-39 01 Vκ2D-40 01 Vκ3D-7 01 Vκ1D-8 01 or d01 Vκ1D-43 01Vκ3D-11 01 or d01 Vκ1D-12 02 Vκ1D-13 d01 Vκ3D-15 d01 Vκ1D-16 01 Vκ1D-1701 Vκ3D-20 01 Vκ2D-26 01 or d01 Vκ2D-28 01 or d01 Vκ2D-29 01 Vκ2D-30 01Vκ1D-33 01 Vκ1D-39 01

In one aspect of Table 12 there no Vκ1D-39 gene segment present in thegenome.

For the avoidance of doubt, any reference to Table 12 herein, includingin the claims, can be read with or without the limitation that in oneaspect of Table 12, there no Vκ1D-39 gene segment present in the genome.

In an alternative Table 12, the Vκ2D-26 allele is Vκ2D-26*d02.

TABLE 13 Igλ Alleles #1-L1 or P1 Alleles ID Allele Cλ7 01 Jλ7 01 Cλ6 04Jλ6 01 Cλ3 03 Jλ3 02 Cλ2 02 Jλ2 01 Cλ1 02 Jλ1 01 Vλ3-1 01

TABLE 14 Igλ Alleles #2-L2 or P2 Alleles ID Allele Cλ7 01 Jλ7 01 Cλ6 04Jλ6 01 Cλ3 03 Jλ3 02 Cλ2 02 Jλ2 01 Cλ1 02 Jλ1 01 Vλ3-1 01 Vλ4-3 01 Vλ2-801 Vλ3-9 01 Vλ3-10 01 Vλ2-11 01 Vλ3-12 02 Vλ2-14 01 Vλ3-16 01 Vλ2-18 01

TABLE 15 Igλ Alleles #3-L3 or P3 Alleles ID Allele Cλ7 01 Jλ7 01 Cλ6 04Jλ6 01 Cλ3 03 Jλ3 02 Cλ2 02 Jλ2 01 Cλ1 02 Jλ1 01 Vλ3-1 01 Vλ4-3 01 Vλ2-801 Vλ3-9 01 Vλ3-10 01 Vλ2-11 01 Vλ3-12 02 Vλ2-14 01 Vλ3-16 01 Vλ2-18 01Vλ3-19 01 Vλ3-21 01 or d01 Vλ3-22 01 Vλ2-23 02 or d02 Vλ3-25 01 or d03Vλ3-27 01

TABLE 16 Igλ Alleles #4-L4 or P4 Alleles ID Allele Cλ7 01 Jλ7 01 Cλ6 04Jλ6 01 Cλ3 03 Jλ3 02 Cλ2 02 Jλ2 01 Cλ1 02 Jλ1 01 Vλ3-1 01 Vλ4-3 01 Vλ2-801 Vλ3-9 01 Vλ3-10 01 Vλ2-11 01 Vλ3-12 02 Vλ2-14 01 Vλ3-16 01 Vλ2-18 01Vλ3-19 01 Vλ3-21 01 or d01 Vλ3-22 01 Vλ2-23 02 or d02 Vλ3-25 01 or d03Vλ3-27 01 Vλ1-36 01 Vλ5-37 01 Vλ5-39 01 Vλ1-40 01 Vλ7-43 01 Vλ1-44 01Vλ5-45 03 Vλ7-46 01

TABLE 17 Igλ Alleles #5-L5 or P5 Alleles ID Allele Cλ7 01 Jλ7 01 Cλ6 04Jλ6 01 Cλ3 03 Jλ3 02 Cλ2 02 Jλ2 01 Cλ1 02 Jλ1 01 Vλ3-1 01 Vλ4-3 01 Vλ2-801 Vλ3-9 01 Vλ3-10 01 Vλ2-11 01 Vλ3-12 02 Vλ2-14 01 Vλ3-16 01 Vλ2-18 01Vλ3-19 01 Vλ3-21 01 or d01 Vλ3-22 01 Vλ2-23 02 or d02 Vλ3-25 01 or d03Vλ3-27 01 Vλ1-36 01 Vλ5-37 01 Vλ5-39 01 Vλ1-40 01 Vλ7-43 01 Vλ1-44 01Vλ5-45 03 Vλ7-46 01 Vλ1-47 01 Vλ9-49 01 Vλ1-51 01 Vλ5-52 01 Vλ10-54 02

TABLE 18 Igλ Alleles #6-L6 or P6 Alleles (a complete repertoire offunctional human lambda alleles) 1.6 ID Allele Cλ7 01 Jλ7 01 Cλ6 04 Jλ601 Cλ3 03 Jλ3 02 Cλ2 02 Jλ2 01 Cλ1 02 Jλ1 01 Vλ3-1 01 Vλ4-3 01 Vλ2-8 01Vλ3-9 01 Vλ3-10 01 Vλ2-11 01 Vλ3-12 02 Vλ2-14 01 Vλ3-16 01 Vλ2-18 01Vλ3-19 01 Vλ3-21 d01 Vλ3-22 01 Vλ2-23 02 or d02 Vλ3-25 01 or d03 Vλ3-2701 Vλ1-36 01 Vλ5-37 01 Vλ5-39 01 Vλ1-40 01 Vλ7-43 01 Vλ1-44 01 Vλ5-45 03Vλ7-46 01 Vλ1-47 01 Vλ9-49 01 Vλ1-51 01 Vλ5-52 01 Vλ10-54 02 Vλ6-57 01Vλ4-60 03 or d03 Vλ8-61 01 Vλ4-69 01

With respect to Table 18, in one aspect there is no Cλ6 or Jλ6 genesegment present in the genome. In another aspect, additionally oralternatively, there is no Vλ3-22 and/or Vλ5-39 and/or Vλ10-54 genesegment.

For the avoidance of doubt, any reference to Table 18 herein, includingin the claims, can be read with or without the limitation that, in oneaspect of Table 18, there no Cλ6 or Jλ6 present, and without or withoutthe limitation that there is no Vλ3-22 and/or Vλ5-39 and/or Vλ10-54 genesegment.

The disclosure of WO2013/041844 is incorporated herein by reference. Theexamples of gene segments in WO2013/041844 are specifically incorporatedherein as though specifically and explicitly disclosed herein aspossible gene segments with respect to the present invention and forpossible inclusion in one or more claims herein.

Each aspect, embodiment, clause or provision described herein can becombined in a non-human vertebrate capable of expressing one or morehuman gene segments disclosed in WO2013/041844 and/or described hereinor a binding site or antibody that is a product of recombination of oneor more human gene segments disclosed in WO2013/041844 and/or describedherein, as appropriate,

The gene segments disclosed in Tables 1-7 of WO2013/041844 arespecifically and explicitly disclosed herein as possible gene segmentsequences with respect to the present invention and for possibleinclusion in one or more claims herein. The sequences are set out in thesequence listing filed with that application.

Further examples of sequences of gene segments for use in the context ofthe invention are set out in the sequences included at the end of thedescription.

In a preferred embodiment, the genome of the vertebrate or cell of theinvention comprises one or more gene segments from any one of Tables 1to 18. In a further preferred embodiment, the genome of the vertebrateor cell of the invention comprises a combination of any two genesegments from any one of Tables 1 to 18. In a yet further preferredembodiment, the genome of the vertebrate or cell of the inventioncomprises a combination of any three gene segments from any one ofTables 1 to 18.

In the most preferred embodiment, the genome of the vertebrate or cellof the invention comprises a combination of any four gene segments fromany one of Tables 1 to 18. Preferably a VH and a JH heavy chain segmentis selected from Table 7 and a VL and a JL light chain segment isselected from Table 12 or Table 18. Optionally a D heavy chain segmentis selected from Table 7.

The invention further relates to a non-human vertebrate (eg, a mouse orrat) or cell whose genome comprises human VL and JL gene segmentsupstream of a constant region at an endogenous light chain locus,wherein the vertebrate or cell expresses immunoglobulin light chainscomprising human variable regions, or where the cell can develop into avertebrate that expresses said light chains, wherein said immunoglobulinlight chains comprise light chains comprising human variable regionsderived from recombination of (i) human Vκ and Jκ gene segments selectedfrom the group consisting of Vκ and Jκ gene segments of any one ofTables 8 to 12 or (ii) human Vλ and Jλ gene segments selected from thegroup consisting of Vλ and Jλ gene segments of any one of Tables 12 to18.

In one embodiment the vertebrate or cell of the invention expresseslight chains comprising human lambda variable regions and wherein atleast 60%, 70%, 80%, 85%, 90% or 95% of the variable regions of suchlight chains are derived from recombination of human Vλ and Jλ genesegments.

Optionally the light chains are expressed as IgG antibodies, for exampleIgG1 or IgG2b, optionally IgG2a.

The constant region can be a kappa or lambda constant region; optionallyhuman, mouse or rat constant region. Said endogenous light chain locuscan be a kappa or lambda locus; optionally wherein the genome comprisesat least the V and J gene segments of Table 8 at an endogenous lightchain locus, for example the kappa locus and/or at least the V and Jgene segments of Table 13 at an endogenous light chain locus, forexample the lambda or kappa locus.

Said light chains can comprise immunoglobulin light chains comprisinghuman variable regions that derived from recombination of (ii), eachsuch variable region being expressed with a constant region encoded by aCλ gene segment selected from the group consisting of the Cλ genesegments of Table 18.

In this embodiment of the invention, the vertebrate or cell can expresslight chains comprising human lambda variable regions and at least 60%,70% or 80% of the variable regions of such light chains can be derivedfrom recombination of human Vλ and Jλ gene segments, for example the Vand J segments listed in Table 18. A invention also relates to anon-human vertebrate (eg, a mouse or rat) or cell whose genome compriseshuman VH, D and JH gene segments upstream of a constant region at anendogenous heavy chain locus, wherein the vertebrate or cell expressesimmunoglobulin heavy chains comprising human variable regions or thecell can develop into a vertebrate that expresses said heavy chains, orwhere the cell can express immunoglobulin heavy chains comprising humanvariable regions, wherein said immunoglobulin heavy chains compriseheavy chains comprising human variable regions derived fromrecombination of (iii) human VH, D and JH gene segments selected fromthe group consisting of VH, D and JH gene segments of Table 7.

In one embodiment said light chains are co-expressed with said heavychains to form antibodies, eg, IgG antibodies.

A non-human vertebrate (eg, a mouse or rat) or cell whose genomecomprises an Ig gene segment repertoire produced by targeted insertionof human Ig gene segments into one or more endogenous Ig loci, thegenome comprising human Vλ and Jλ gene segments upstream of a constantregion, wherein the human Vλ and Jλ gene segments have been provided byinsertion into an endogenous light chain locus of the vertebrate orcell, wherein the vertebrate comprises immunoglobulin light chainscomprising lambda variable regions (lambda light chains) or the cell candevelop into a vertebrate that expresses said immunoglobulin lightchains, wherein the lambda light chains comprise immunoglobulin lightchains comprising lambda variable regions derived from recombination ofhuman Vλ and Jλ gene segments; wherein at least 60%, 70%, 80% or 90% ofthe variable regions of the lambda light chains expressed by thevertebrate are derived from recombination of human Vλ and Jλ genesegments; optionally wherein the vertebrate or cell any vertebrate orcell disclosed herein.

A variable domain or region derived from, or produced as a result of,recombination of human gene segments is also referred to herein as arecombinant of said gene segments.

The gene segments in the heavy and/or light locus are operably linked tothe constant region, so that the vertebrate is capable of producing anantibody heavy or light chain produced by recombination of the genesegments.

In one embodiment the cell or non-human vertebrate has a genomecomprising the kappa segments of table 8, or table 9 or table 10, table11 or table 12, or any combination thereof.

In one embodiment the cell or non-human vertebrate has a genomecomprising the lambda segments of table 13 or table 14 or table 15 ortable 16 or table 17 or table 18, or any combination thereof.

In one embodiment the cell or non-human vertebrate has a genomecomprising the heavy chain segments of table 1 or table 2 or table 3 ortable 4 or table 5 or table 6 or table 7 or any combination thereof.

In one embodiment the cell or non-human vertebrate of the invention canexpress light chains comprising human variable regions derived fromrecombination (i.e. recombinants) of (i) human Vκ and Jκ gene segmentsselected from the group consisting of Vκ and Jκ gene segments of Table8, or table 9, or table 10, or table 11 or table 12, or any combinationthereof.

In one embodiment the cell or non-human vertebrate of the invention canexpress light chains comprising human variable regions derived fromrecombination (i.e. recombinants) of (ii) human Vλ and Jλ gene segmentsselected from the group consisting of Vλ and Jλ gene segments of Table13 or table 14 or table 15 or table 16 or table 17 or table 18, or anycombination thereof.

In one embodiment the cell or vertebrate of the invention can expressheavy chains comprising human variable regions derived fromrecombination (i.e. recombinants) of human VH, D and JH gene segmentsselected from the group consisting of VH, D and JH gene segments oftable 1 or table 2, or table 3, or table 4 or table 5 or table 6 ortable 7, or any combination thereof.

The invention further includes the following provisions

Provision 1—The vertebrate or cell of the invention has a genomecomprising one or more alleles selected from alleles numbered 1 to 68 inTable 7.

Provision 2—The vertebrate or cell according to provision 1 comprisingallele number 1 from Table 1 and one or more alleles selected fromalleles numbered 2 to 68 of Table 7.

Provision 3—The vertebrate or cell according to any preceding provisioncomprising allele number 2 from Table 7 and one or more alleles selectedfrom alleles numbered 3 to 68 of Table 7.

Provision 4—The vertebrate or cell according to any preceding provisioncomprising allele number 3 from Table 7 and one or more alleles selectedfrom alleles numbered 4 to 68 of Table 7.

Provision 5—The vertebrate or cell according to any preceding provisioncomprising allele number 4 from Table 7 and one or more alleles selectedfrom alleles numbered 5 to 68 of Table 7.

Provision 6—The vertebrate or cell according to any preceding provisioncomprising allele number 5 from Table 7 and one or more alleles selectedfrom alleles numbered 6 to 68 of Table 7.

Provision 7—The vertebrate or cell according to any preceding provisioncomprising allele number 6 from Table 7 and one or more alleles selectedfrom alleles numbered 7 to 68 of Table 7.

Provision 8—The vertebrate or cell according to any preceding provisioncomprising allele number 7 from Table 7 and one or more alleles selectedfrom alleles numbered 8 to 68 of Table 7.

Provision 9—The vertebrate or cell according to any preceding provisioncomprising allele number 8 from Table 7 and one or more alleles selectedfrom alleles numbered 9 to 68 of Table 7.

Provision 10—The vertebrate or cell according to any preceding provisioncomprising allele number 9 from Table 7 and one or more alleles selectedfrom alleles numbered 10 to 68 of Table 7.

Provision 11—The vertebrate or cell according to any preceding provisioncomprising allele number 10 from Table 7 and one or more allelesselected from alleles numbered 11 to 68 of Table 7.

Provision 12—The vertebrate or cell according to any preceding provisioncomprising allele number 11 from Table 7 and one or more allelesselected from alleles numbered 12 to 68 of Table 7.

Provision 13—The vertebrate or cell according to any preceding provisioncomprising allele number 12 from Table 7 and one or more allelesselected from alleles numbered 13 to 68 of Table 7.

Provision 14—The vertebrate or cell according to any preceding provisioncomprising allele number 13 from Table 7 and one or more allelesselected from alleles numbered 14 to 68 of Table 7.

Provision 15—The vertebrate or cell according to any preceding provisioncomprising allele number 14 from Table 7 and one or more allelesselected from alleles numbered 15 to 68 of Table 7.

Provision 16—The vertebrate or cell according to any preceding provisioncomprising allele number 15 from Table 7 and one or more allelesselected from alleles numbered 16 to 68 of Table 7.

Provision 17—The vertebrate or cell according to any preceding provisioncomprising allele number 16 from Table 7 and one or more allelesselected from alleles numbered 17 to 68 of Table 7.

Provision 18—The vertebrate or cell according to any preceding provisioncomprising allele number 17 from Table 7 and one or more allelesselected from alleles numbered 18 to 68 of Table 7.

Provision 19—The vertebrate or cell according to any preceding provisioncomprising allele number 18 from Table 7 and one or more allelesselected from alleles numbered 19 to 68 of Table 7.

Provision 20—The vertebrate or cell according to any preceding provisioncomprising allele number 19 from Table 7 and one or more allelesselected from alleles numbered 20 to 68 of Table 7.

Provision 21—The vertebrate or cell according to any preceding provisioncomprising allele number 20 from Table 7 and one or more allelesselected from alleles numbered 21 to 68 of Table 7.

Provision 22—The vertebrate or cell according to any preceding provisioncomprising allele number 21 from Table 7 and one or more allelesselected from alleles numbered 22 to 68 of Table 7.

Provision 23—The vertebrate or cell according to any preceding provisioncomprising allele number 22 from Table 7 and one or more allelesselected from alleles numbered 23 to 68 of Table 7.

Provision 24—The vertebrate or cell according to any preceding provisioncomprising 4allele number 23 from Table 7 and one or more allelesselected from alleles numbered 24 to 68 of Table 7.

Provision 25—The vertebrate or cell according to any preceding provisioncomprising allele number 24 from Table 7 and one or more allelesselected from alleles numbered 25 to 68 of Table 7.

Provision 26—The vertebrate or cell according to any preceding provisioncomprising allele number 25 from Table 7 and one or more allelesselected from alleles numbered 26 to 68 of Table 7.

Provision 27—The vertebrate or cell according to any preceding provisioncomprising allele number 26 from Table 7 and one or more allelesselected from alleles numbered 27 to 68 of Table 7.

Provision 28—The vertebrate or cell according to any preceding provisioncomprising allele number 27 from Table 7 and one or more allelesselected from alleles numbered 28 to 68 of Table 7.

Provision 29—The vertebrate or cell according to any preceding provisioncomprising allele number 28 from Table 7 and one or more allelesselected from alleles numbered 29 to 68 of Table 7.

Provision 30—The vertebrate or cell according to any preceding provisioncomprising allele number 29 from Table 7 and one or more allelesselected from alleles numbered 30 to 68 of Table 7.

Provision 31—The vertebrate or cell according to any preceding provisioncomprising allele number 30 from Table 7 and one or more allelesselected from alleles numbered 31 to 68 of Table 7.

Provision 32—The vertebrate or cell according to any preceding provisioncomprising allele number 31 from Table 7 and one or more allelesselected from alleles numbered 32 to 68 of Table 7.

Provision 33—The vertebrate or cell according to any preceding provisioncomprising allele number 32 from Table 7 and one or more allelesselected from alleles numbered 33 to 68 of Table 7.

Provision 34—The vertebrate or cell according to any preceding provisioncomprising allele number 33 from Table 7 and one or more allelesselected from alleles numbered 34 to 68 of Table 7.

Provision 35—The vertebrate or cell according to any preceding provisioncomprising allele number 34 from Table 7 and one or more allelesselected from alleles numbered 35 to 68 of Table 7.

Provision 36—The vertebrate or cell according to any preceding provisioncomprising allele number 35 from Table 7 and one or more allelesselected from alleles numbered 36 to 68 of Table 7.

Provision 37—The vertebrate or cell according to any preceding provisioncomprising allele number 36 from Table 7 and one or more allelesselected from alleles numbered 37 to 68 of Table 7.

Provision 38—The vertebrate or cell according to any preceding provisioncomprising allele number 37 from Table 7 and one or more allelesselected from alleles numbered 38 to 68 of Table 7.

Provision 39—The vertebrate or cell according to any preceding provisioncomprising allele number 38 from Table 7 and one or more allelesselected from alleles numbered 39 to 68 of Table 7.

Provision 40—The vertebrate or cell according to any preceding provisioncomprising allele number 39 from Table 7 and one or more allelesselected from alleles numbered 40 to 68 of Table 7.

Provision 41—The vertebrate or cell according to any preceding provisioncomprising allele number 40 from Table 7 and one or more allelesselected from alleles numbered 41 to 68 of Table 7.

Provision 42—The vertebrate or cell according to any preceding provisioncomprising allele number 41 from Table 7 and one or more allelesselected from alleles numbered 42 to 68 of Table 7.

Provision 43—The vertebrate or cell according to any preceding provisioncomprising allele number 42 from Table 7 and one or more allelesselected from alleles numbered 43 to 68 of Table 7.

Provision 44—The vertebrate or cell according to any preceding provisioncomprising allele number 43 from Table 7 and one or more allelesselected from alleles numbered 44 to 68 of Table 7.

Provision 45—The vertebrate or cell according to any preceding provisioncomprising allele number 44 from Table 7 and one or more allelesselected from alleles numbered 45 to 68 of Table 7.

Provision 46—The vertebrate or cell according to any preceding provisioncomprising allele number 45 from Table 7 and one or more allelesselected from alleles numbered 46 to 68 of Table 7.

Provision 47—The vertebrate or cell according to any preceding provisioncomprising allele number 46 from Table 7 and one or more allelesselected from alleles numbered 47 to 68 of Table 7.

Provision 48—The vertebrate or cell according to any preceding provisioncomprising allele number 47 from Table 7 and one or more allelesselected from alleles numbered 48 to 68 of Table 7.

Provision 49—The vertebrate or cell according to any preceding provisioncomprising allele number 48 from Table 7 and one or more allelesselected from alleles numbered 49 to 68 of Table 7.

Provision 50—The vertebrate or cell according to any preceding provisioncomprising allele number 49 from Table 7 and one or more allelesselected from alleles numbered 50 to 68 of Table 7.

Provision 51—The vertebrate or cell according to any preceding provisioncomprising allele number 50 from Table 7 and one or more allelesselected from alleles numbered 51 to 68 of Table 7.

Provision 52—The vertebrate or cell according to any preceding provisioncomprising allele number 51 from Table 7 and one or more allelesselected from alleles numbered 52 to 68 of Table 7.

Provision 53—The vertebrate or cell according to any preceding provisioncomprising allele number 52 from Table 7 and one or more allelesselected from alleles numbered 53 to 68 of Table 7.

Provision 54—The vertebrate or cell according to any preceding provisioncomprising allele number 53 from Table 7 and one or more allelesselected from alleles numbered 54 to 68 of Table 7.

Provision 55—The vertebrate or cell according to any preceding provisioncomprising allele number 54 from Table 7 and one or more allelesselected from alleles numbered 55 to 68 of Table 7.

Provision 56—The vertebrate or cell according to any preceding provisioncomprising allele number 56 from Table 7 and one or more allelesselected from alleles numbered 57 to 68 of Table 7.

Provision 57—The vertebrate or cell according to any preceding provisioncomprising allele number 57 from Table 7 and one or more allelesselected from alleles numbered 58 to 68 of Table 7.

Provision 58—The vertebrate or cell according to any preceding provisioncomprising allele number 58 from Table 7 and one or more allelesselected from alleles numbered 59 to 68 of Table 7.

Provision 59 The vertebrate or cell according to any preceding provisioncomprising allele number 59 from Table 7 and one or more allelesselected from alleles numbered 60 to 68 of Table 7.

Provision 60—The vertebrate or cell according to any preceding provisioncomprising allele number 60 from Table 7 and one or more allelesselected from alleles numbered 61 to 68 of Table 7.

Provision 61—The vertebrate or cell according to any preceding provisioncomprising allele number 61 from Table 7 and one or more allelesselected from alleles numbered 62 to 68 of Table 7.

Provision 62—The vertebrate or cell according to any preceding provisioncomprising allele number 62 from Table 7 and one or more allelesselected from alleles numbered 63 to 68 of Table 7.

Provision 63—The vertebrate or cell according to any preceding provisioncomprising allele number 63 from Table 7 and one or more allelesselected from alleles numbered 64 to 68 of Table 7.

Provision 64—The vertebrate or cell according to any preceding provisioncomprising allele number 64 from Table 7 and one or more allelesselected from alleles numbered 65 to 68 of Table 7.

Provision 65—The vertebrate or cell according to any preceding provisioncomprising allele number 65 from Table 7 and one or more allelesselected from alleles numbered 66 to 68 of Table 7.

Provision 66—The vertebrate or cell according to any preceding provisioncomprising allele number 66 from Table 7 and one or more allelesselected from alleles numbered 67 or 68 of Table 7.

Provision 67—The vertebrate or cell according to any preceding provisioncomprising allele number 67 from Table 7 and allele number 68 from Table7.

In an example, the vertebrate or cell according to any precedingprovision comprises one or more or all JH alleles from Table 7, eg,JH2*02 and/or at least JH6*02 (which is useful for producing long HCDR3V domains for human therapeutic use as shown in the examples).

Thus, each allele combined with any other allele in Table 7 isexplicitly disclosed herein. The same structure of combinations isdisclosed in relation to the alleles of Table 12 and Table 18.Therefore, each allele in Table 7 combined with any other allele inTable 12 or Table 18 is disclosed, and each allele in Table 12 isdisclosed in combination with every other allele in Table 12, and eachallele in Table 18 is disclosed in combination with every other allelein Table 18.

In an example, the vertebrate or cell according to any precedingprovision comprises one or more or all Jκ alleles from Table 12, eg, atleast Jκ2*01 and/or Jκ4*01 (which is useful for producing Vκ domains forhuman therapeutic use as shown in the examples).

In one embodiment, the cell or vertebrate of the invention has a genomecomprising the gene segments VH3-23*04, JH2*01, Vκ4-1*01 and/or Jκ2*01.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the gene segments VH3-7*01, JH6*02, Vκ2-28*01 and/orJκ4*01.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the gene segments VH7-4-1*01, JH6*02, Vκ2-28*01 and/orJκ4*01. Optionally the genome further comprises D3-16*02.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the gene segments VH4-4-1*02, JH6*02, Vκ1D-13*01and/or Jκ4*01. Optionally the genome further comprises D3-10*01.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the gene segments VH1-3*01, JH6*02, Vκ1-12*01 and/orJκ4*01. Optionally the genome further comprises D3-10*01.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the gene segments VH3-13*01, JH6*02, Vκ1D-12*02 and/orJκ4*01. Optionally the genome further comprises D3-9*01.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the gene segments VH4-4*02, JH6*02, Vκ1D-13*01 and/orJκ4*01. Optionally the genome further comprises D3-10*01.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the gene segments VH3-13*01, JH6*02, Vκ3-20*01 and/orJκ4*01. Optionally the genome further comprises D3-10*01.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the gene segments VH3-23*04, JH6*02, VK1-17*01 and/orJK4*01. Optionally the genome further comprises D3-22*01.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the gene segments VH3-7*01, JH6*02, VK1D-39*01 and/orJK4*01. Optionally the genome further comprises D3-9*01.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the gene segments VH3-13*01, JH6*02, VK1D-39*01 and/orJK4*01. Optionally the genome further comprises D3-10*01.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the gene segments VH3-13*01, JH6*02, VK3-11*01 and/orJK4*01. Optionally the genome further comprises D3-10*01.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the gene segments VH4-4*02, JH6*02, VK1D-16*01 and/orJK4*01. Optionally the genome further comprises D3-9*01.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the gene segments VH3-20*d01, JH6*02, VK1-9*d01 and/orJK4*01. Optionally the genome further comprises D3-10*01.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the human gene segment VH3-23*04. Additionally oralternatively, heavy chain variable domains of the antibody of theinvention are encoded by (i) human VH3-23*04, D and JH segments.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the human gene segment VH3-9*01. Additionally oralternatively, heavy chain variable domains of the antibody of theinvention are encoded by (i) human VH3-9*01, D and JH segments.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the human gene segment Vκ1-12*02 or Vκ1D-12*02.Additionally or alternatively, light chain variable domains of theantibody of the invention are encoded by (i) human Vκ1-12*02 orVκ1D-12*02 and Jκ segments.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the human gene segment Vκ2-28*01. Additionally oralternatively, light chain variable domains of the antibody of theinvention are encoded by (i) human Vκ2-28*01 and Jκ segments.

In a further embodiment, the cell or vertebrate of the invention has agenome comprising the human gene segment Vκ4-1*01. Additionally oralternatively, light chain variable domains of the antibody of theinvention are encoded by (i) human Vκ4-1*01 and Jκ segments. In afurther embodiment, the cell or vertebrate of the invention has a genomecomprising the combination of gene segments described above with JH6*01in place of JH6*02.

In all embodiments described herein, the heavy chain V and J region canoptionally be recombined with each other and with a D region definedherein to form a heavy chain variable domain. In addition, the lightchain V and J regions can optionally be recombined to form a light chainvariable domain.

The invention extends to an antibody or antigen binding fragmentcomprising human variable domains produced or derived from recombinationof any of the above combinations of gene segments.

Antibodies or fragments according to the invention are shown in theexamples to be useful for producing productive gene segmentrecombination in vivo, which display junctional mutation and somaticmutation, and produce domains that can specifically bind antigen withgood binding kinetics.

The invention includes antibodies or antigen binding fragments thereofthat are obtained or obtainable by recombination, in vivo in a mouse,mammal or vertebrate of the invention following immunisation, of one ormore D gene segments, one or more VH gene segments and one or more ofthe human JH gene segments J_(H)2*01 and J_(H)6*02.

In one embodiment, the cell or vertebrate of the invention can expressheavy chains comprising human variable regions derived fromrecombination of one or more D gene segments, one or more J_(H) genesegments and one or more of the following VH gene segmentsV_(H)3-20*d01, VH1-24*d01 and V_(H)2-26*d01.

Therefore, the invention includes antibodies or antigen bindingfragments thereof that are obtained or obtainable by recombination, invivo in a mouse, mammal or vertebrate of the invention followingimmunisation, of one or more D gene segments, one or more J_(H) genesegments and one or more of the human VH gene segments V_(H)3-20*d01,V_(H)1-24*d01 and V_(H)2-26*d01.

In a further embodiment, the cell or vertebrate of the invention canadditionally or alternatively express light chains comprising humanvariable regions derived from recombination of one or more Jκ genesegments and one or more of the human Vκ gene segments Vκ5-2*d01,Vκ1-9*d01, Vκ1D-8*d01, Vκ3D-11*d01, Vκ1D-13*d01, Vκ3D-15*d01,Vκ2D-26*d01 and Vκ2D-28*d01 or recombination of one or more Jλ genesegments and one or more of the human Vλ gene segments Vλ2-22*d01,Vλ2-23*d02, Vλ3-25*d03 and Vλ4-60*d03.

Therefore, the invention includes antibodies or antigen bindingfragments thereof that are obtained or obtainable by recombination, invivo in a mouse, mammal or vertebrate of the invention followingimmunisation, of one or more Jκ gene segments and one or more of thehuman Vκ gene segments Vκ5-2*d01, Vκ1-9*d01, Vκ1D-8*d01, Vκ3D-11*d01,Vκ2D-26*d01 and Vκ2D-28*d01 or of one or more Jλ gene segments and oneor more of the human Vλ gene segments Vλ2-22*d01, Vλ2-23*d02, Vλ3-25*d03and Vλ4-60*d03.

In all aspects of the invention, the cell can be a hybridoma cell, a Bcell, optionally an immortalised B cell, or an embryonic stem cell.

In one embodiment the vertebrate or cell comprises a constant regionthat is a kappa or lambda constant region; optionally a mouse or ratconstant region. In one embodiment the constant region may be a humanconstant region.

In one embodiment the vertebrate or cell disclosed herein comprises anendogenous light chain locus which is a kappa or lambda locus;optionally wherein the genome comprises at least the V and J genesegments of Table 8, 9 or 10 at an endogenous light chain locus, eg thekappa locus and/or at least the V and J gene segments of Table 13 or 14at an endogenous light chain locus, eg either the lambda or kappa locus.

In one embodiment the vertebrate or cell disclosed herein comprisescomprise immunoglobulin light chains comprising human variable regionsthat are derived from recombination of human Vλ and Jλ gene segmentsselected from the group consisting of Vλ and Jλ gene segments of Table18, each such variable region being expressed with a constant regionencoded by a Cλ gene segment selected from the group consisting of theCλ gene segments of Table 18.

In one embodiment the light chains comprise human variable regionsderived from recombination of human Vλ and Jλ gene segments selectedfrom the group consisting of Vλ and Jλ gene segments of Table 18.

In all embodiments of the invention, the VH and VL domains canoptionally form an antigen binding site.

The cell of any aspect of the invention can be a hybridoma cell or a Bcell, optionally an immortalised B cell. The cell can also be an EScell. The ES cell can be part of a population of at least 90, 150 ormore than 200 cells. In an example, the population of cells is containedon one or more multi-well plates (eg, 96-well plates) and may, forexample, be a sorted population where single cells are comprised bydifferent, respective wells of a plate. The vertebrate of any aspect canbe comprised within a container comprising filtered air, optionallycomprising an air filter.

In an embodiment, the container inner environment is sterile, eg, aspossible using a standard animal container, eg, a Techniplast™ mouseloft (http://www.tecniplast.it/us/product/mouse-loft.html). In anexample, the container has a plastic body, eg, a translucent ortransparent body.

The container comprising the vertebrates may have a volume of no morethan four, 3, 2 or 1 metres³. The container can comprise a plurality ofvertebrates, eg, a male and a female (eg, a fertile pair).

In one embodiment the vertebrates of the invention are at least 3.5weeks old, eg 4 weeks, 5 weeks, 6 weeks, 7 weeks old.

The use of the gene segments as claimed, specifically as indicated intables 1-18, provides for the advantages seen in the Examples.

The invention further relates to a method of producing an antibody or anantigen binding fragment thereof, the method comprising immunising avertebrate of the invention with an antigen and recovering the antibodyor fragment or recovering a cell producing the antibody or fragment;optionally modifying the isolated antibody or fragment so that itcomprises human constant regions.

The invention also includes a method of producing an antibody or anantigen binding fragment thereof, the method comprising isolating anantibody or antigen binding fragment thereof from a cell of theinvention; and optionally modifying the isolated antibody or fragment sothat it comprises human constant regions.

Recombinant DNA technology can be used to produce a modified nucleotidesequence encoding the modified antibody or fragment.

The method may comprise

(a) isolating from the vertebrate a B-cell encoding an antibody thatbinds the antigen,(b) identifying or copying a nucleotide sequence of the B-cell thatencodes a VH domain of the antibody and/or identifying or copyingnucleotide sequence of the B-cell that encodes a VL domain of theantibody; and(c) using the sequence(s) to produce an isolated antibody comprising theVH and/or VL domain; optionally wherein the isolated antibody compriseshuman constant regions. The invention includes a method for producing afully humanised antibody comprising immunizing a vertebrate as disclosedherein and then replacing the non-human vertebrate constant region of anantibody specifically reactive with the antigen with a human constantregion, suitably by engineering of the nucleic acid encoding theantibody.

The invention also relates to a humanised antibody produced according toany methods disclosed herein and use of a humanised antibody so producedin medicine.

In a further embodiment, the invention includes isolating an IgG1, IgG2band/or IgM antibody that specifically binds the target antigen.

An isolated antibody of the invention may be produced by expression froma host cell selected from a CHO, HEK293, Cos or yeast (eg, Picchia)cell. In a preferred embodiment, the antibody is an antibody produced byexpression from a CHO cell.

In a further embodiment the isolated antibody or fragment may beformulated with a diluent, carrier, excipient or a drug to produce apharmaceutical composition for human medical use. Optionally theformulated antibody may be packaged in a sterile container, for example,a vial, tube, IV bag or syringe, further optionally producing a kitcomprising combining the package with a label or instructions indicatinguse of the antibody composition for human medical use; optionallywherein the label or instructions comprises a medicament batch numberand/or a marketing authorisation number, further optionally an EMA orFDA marketing authorisation number. In an example, the isolated antibody(eg, produced by a CHO cell) (i) is formulated with a diluent, carrier,excipient or a drug to produce a pharmaceutical composition for humanmedical use, (ii) the formulated antibody is packaged in a sterilecontainer combined with a label or instructions indicating use of theantibody composition for human medical use; wherein the label orinstructions comprises a medicament batch number and/or a marketingauthorisation number (eg, an EMA or FDA marketing authorisation number).In an embodiment of such an example, the antibody specically binds humanPCSK9 and the use is for treating or preventing hyperlipidaemia or forreducing cholesterol in a human. In an embodiment of such an example,the antibody specically binds human IL6Ra and the use is for treating orpreventing an inflammatory condition or rheumatoid arthritis in a human.In an embodiment of such an example, the antibody specically binds humanIL4Ra and the use is for treating or preventing an atopic disease,atopic dermatitis or asthma in a human.

The invention relates to the antibody or fragment produced by the methodof the invention for human medical use and to use of the isolatedantibody or fragment produced by the method of the invention in themanufacture of a medicament for human medical use.

The medicament may be a composition or kit disclosed herein.

The invention includes antibodies and antigen binding fragments thereofcomprising human variable domains produced by or derived fromrecombination of any combination of gene segments disclosed herein,optionally wherein the antibody and fragments are obtained or obtainableby recombination, in vivo in a mouse, mammal or other vertebrate of theinvention following immunisation.

In one embodiment the immunoglobulin heavy chains expressed by the cellor vertebrate are essentially exclusively said heavy chains comprisinghuman variable regions; and said heavy chains comprising human variableregions are expressed as part of serum IgG antibodies, optionally IgG1,IgG2a or IgG2b, or IgM antibodies.

In one embodiment, the cell or vertebrate of the invention expresses anIgG antibody, optionally a IgG1, IgG2a or IgG2b antibody, or a IgMantibody comprising heavy chains as defined herein, wherein the antibodyspecifically binds a target antigen.

The invention further relates to:

The antibody, optionally, produced by a method of the inventioncomprising

(a) a human heavy chain variable domain derived from recombination ofhuman VH, D and JH gene segments selected from the group consisting ofVH, D and JH gene segments of any one of Tables 1-7; and(b) a human light chain variable domain derived from recombination ofhuman V and J gene segments both selected from the V and J gene segmentsof any one of Tables 8-18.

An isolated antibody or antigen binding fragment thereof, optionallyobtained or obtainable by the method of the invention, wherein avariable region of the antibody or fragment comprises one or more mouseor rat activation-induced deaminase (AID) pattern somatic mutationsand/or mouse or rat terminal deoxynucleotidyl transferase (TdT) patternjunctional mutations.

The variable heavy or light domain or region in accordance withinvention can comprises up to 10, including 1, 2, 3, 4, 5, 6, 7, 8, or 9junctional mutations.

In a further embodiment, the variable heavy or light domain or region inaccordance with invention can additionally or alternatively comprise upto 9, including 1, 2, 3, 4, 5, 6, 7, 8, or 8, including 1, 2, 3, 4, 5, 6or 7, somatic mutations.

In a further embodiment, the invention includes an isolated antibody orantigen binding fragment thereof, optionally obtained or obtainable bythe method of the invention, that binds a gamma receptor, and furtheroptionally a human constant light chain. In one embodiment, the Fccomprises human gamma constant domains, e.g. human IgG1, IgG2, IgG3 orIgG4 constant domains.

An isolated antibody or antigen binding fragment thereof, optionallyobtained or obtainable by the method of the invention, wherein theantibody comprises CHO, HEG293, Cos or yeast (eg, Picchia) cellglycosylation.

An antibody or fragment of the invention can specifically bind a humanenzyme.

An antibody or fragment of the invention can specifically bind the humantargets: proprotein convertase PC9, proprotein convertase subtilisinkexin-9 (PCSK9), CD126, IL-4, IL-4 receptor, IL-6, IL-6 receptor, IL-13,IL-18 receptor, Erbb3, cell ASIC1, ANG2, GDF-8, angiopoietin ligand-2,delta-like protein ligand 4, immunoglobulin G1, PDGF ligand, PDGFreceptor or NGF receptor, toxin A or toxin B of Clostridium difficile,relaxin, CD48, Cd20, glucagon receptor, protease activated receptor 2,TNF-Like ligand 1A (TL1A), angiopoietin related-2 (AR-2),angiopoietin-like protein 4, RANKL, angiopoietin-like protein 3(ANGPTL3), delta-like ligand 4 (DLL4), big endothelin-1 (ET-1), activinA, receptor tyrosine kinases, for example human AR-1 and tyrosine kinasewith Ig and EGF homology domains (TIE) or TIE-2 receptor. In an example,the target is PCSK9. In an example, the target is IL-6 receptor (eg,IL6Ra). In an example, the target is IL-4 receptor (eg, IL4Ra).

Preferred aspects of the invention include:

The use of the antibody or fragment thereof in the manufacture of amedicament for use to attenuate or inhibit an IL-4Ra-mediated disease ordisorder in a human. IL-4Ra-mediated or related disorders which aretreated by the ligand, antibody or fragment of the invention include,for example, arthritis (including septic arthritis), herpetiformis,chronic idiopathic urticaria, scleroderma, hypertrophic scarring,Whipple's Disease, benign prostate hyperplasia, lung disorders, such asmild, moderate or severe asthma, inflammatory disorders such asinflammatory bowel disease, allergic reactions, Kawasaki disease, sicklecell disease, Churg-Strauss syndrome, Grave's disease, pre-eclampsia,Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmunehemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis,and nephrosis.

Further IL-4Ra-mediated or related disorders which are treated by theligand, antibody or fragment of the invention include, for example,asthma, COPD (eg, chronic bronchitis, small airway disease oremphysema), inflammatory bowel disease, a fibrotic condition (eg,systemic sclerosis, pulmonary fibrosis, parasite-induced liver fibrosis,or cystic fibrosis), allergy (for example atopic dermatitis, dust miteallergy, pet allergy or food allergy), transplation therapy to preventtransplant rejection, suppression of a delayed-type hypersensitivity ora contact hypersensitivity reaction, as an adjuvant to allergyimmunotherapy or as a vaccine adjuvant.

Further encompassed by the invention is the use of the antibody orfragment thereof in the manufacture of a medicament for prevention ortreatment of a IL-4Ra-mediated or related disorder, wherein said diseaseor condition is an inflammatory disease or condition; an atopic diseaseor condition; a respiratory disease or condition; a disease or conditionassociated with elevated IgE; or a disease or condition associated withelevated IL-4 and/or IL-13 activity.

Further encompassed by the invention is the use of the antibody orfragment thereof in the manufacture of a medicament for prevention ortreatment of a IL-4Ra-mediated or related disease or condition, whereinsaid disease or condition is selected from the group consisting of anairway inflammatory disease or condition, chronic obstructive pulmonarydisease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonaryinfiltrate with eosinophilia, environmental lung disease, pneumonia,bronchiectasis, cystic fibrosis, interstitial lung disease, primarypulmonary hypertension, pulmonary thromboembolism, disorders of thepleura, disorders of the mediastinum, disorders of the diaphragm,hypoventilation, hyperventilation, sleep apnea, acute respiratorydistress syndrome, mesothelioma, sarcoma, graft rejection, graft versushost disease, lung cancer, allergic rhinitis, allergy, asbestosis,aspergilloma, aspergillosis, bronchiectasis, chronic bronchitis,emphysema, eosinophilic pneumonia, idiopathic pulmonary fibrosis,invasive pneumococcal disease, influenza, nontuberculous mycobacteria,pleural effusion, pneumoconiosis, pneumocytosis, pneumonia, pulmonaryactinomycosis, pulmonary alveolar proteinosis, pulmonary anthrax,pulmonary edema, pulmonary embolus, pulmonary inflammation, pulmonaryhistiocytosis X, pulmonary hypertension, pulmonary nocardiosis,pulmonary tuberculosis, pulmonary veno-occlusive disease, rheumatoidlung disease, sarcoidosis, and Wegener's granulomatosis.

Further encompassed by the invention is an anti-IL-4Ra antibody orfragment thereof, as disclosed herein, for prevention or treatment ofany disease or disorder disclosed herein.

Further encompassed by the invention is the use of an anti-IL-4Raantibody or fragment thereof, in a method of medical treatment of adisease or disorder disclosed herein.

Further encompassed by the invention is the use of the ligand, antibodyor fragment of the invention in the manufacture of a medicament for useto attenuate or inhibit a PCSK9-mediated disease or disorder in a human.Non-limiting examples of such diseases or conditions can include, forexample, a lipid disorder, hyperlipoproteinemia, hyperlipidemia;dyslipidemia; hypercholesterolemia, a heart attack, a stroke, coronaryheart disease, atherosclerosis, peripheral vascular disease,claudication, type II diabetes, high blood pressure, and acardiovascular disease or condition.

Further encompassed by the invention is an anti-PCSK9 antibody orfragment thereof, as disclosed herein, for prevention or treatment ofany disease or disorder disclosed herein.

Further encompassed by the invention is the use of an anti-PSCK9antibody or fragment thereof, in a method of medical treatment of adisease or disorder disclosed herein.

Further encompassed by the invention is the use of the ligand, antibodyor fragment of the invention in the manufacture of a medicament for useto attenuate or inhibit an IL-6Ra-mediated disease or disorder in ahuman.

Said IL-6Ra-mediated disease or condition can be an inflammatory diseaseor condition. Said IL-6Ra-mediated disease or condition can be selectedfrom the group consisting of an inflammatory bowel disease (IBD),Crohn's disease, rheumatoid arthritis, psoriasis, bronchiolitis,gingivitis, transplant rejection, allogenic transplant rejection,graft-versus-host disease (GvHD), asthma, adult respiratory distresssyndrome (ARDS), septic shock, ulcerative colitis, Sjorgen's syndrome,airway inflammation, Castleman's disease, periodontitis, atopicdermatitis, systemic lupus erythematosus and coronary heart disease.

Further encompassed by the invention is an anti-IL-6Ra antibody orfragment thereof, as disclosed herein, for prevention or treatment ofany disease or disorder disclosed herein.

Further encompassed by the invention is the use of an anti-IL-6Raantibody or fragment thereof, in a method of medical treatment of adisease or disorder disclosed herein.

In a further embodiment, the invention relates to the following aspects:

-   -   1. A non-human vertebrate or vertebrate cell (eg, a mouse or        rat) whose genome comprises human JH2*01 and/or human JH6*02,        one or more human VH gene segments and one or more human D gene        segments upstream of a constant region at an endogenous heavy        chain locus and/or human Jκ2*01 and/or human Jκ4*01 and one or        more human Vκ gene segments upstream of a constant region at an        endogenous light chain locus, wherein the gene segments in each        locus are operably linked to the constant region thereof so that        the cell or vertebrate is capable of producing an antibody heavy        chain and an antibody light chain, or where the cell can develop        into a vertebrate that expresses an antibody heavy chain and an        antibody light chain, wherein the heavy chain is produced by        recombination of the human JH2*01 and/or JH6*02 segment with a D        segment and a VH segment and/or the antibody light chain is        produced by recombination of the human Jκ2*01 and/or Jκ4*01        segment with a Vκ segment        -   wherein said one or more human VH gene segments of the heavy            chain locus comprise or consist of one, more or all human VH            gene segments selected from the group consisting of            VH3-23*04, VH7-4-1*01, VH4-4*02, VH1-3*01, VH3-13*01,            VH3-7*01 and VH3-20*d01        -   or        -   wherein said one or more human Vκ gene segments comprise or            consist of one, more or all human VH gene segments selected            from the group consisting of Vκ4-1*01, Vκ2-28*01,            Vκ1D-13*d01, Vκ1-12*01, Vκ1D-12*02, Vκ3-20*01, Vκ1-17*01,            Vκ1D-39*01, Vκ3-11*01, Vκ1D-16*01 and Vκ1-9*d01.        -   Optionally the human gene segment is the recombined form of            the gene segment that includes one or more mutations            relative to the germline human gene segment sequence    -   2. The cell or vertebrate of aspect 1 wherein the genome        comprises human J_(H)2*01 and/or human JH6*02, one or more human        VH gene segments and one or more human D gene segments upstream        of a constant region at an endogenous heavy chain locus and or        human Jκ2*01 and/or human Jκ4*01 and one or more human Vκ gene        segments upstream of a constant region at an endogenous light        chain locus,    -   3. The cell or vertebrate of aspect 2 wherein said one or more        human VH gene segments of the heavy chain locus comprise or        consist of one, more or all human VH gene segments selected from        the group consisting of VH3-23*04, VH7-4-1*01, VH4-4*02,        VH1-3*01, VH3-13*01, VH3-7*01 and VH3-20*d01 and wherein said        one or more human Vκ gene segments comprise or consist of one,        more or all human VH gene segments selected from the group        consisting of Vκ4-1*01, Vκ2-28*01, Vκ1D-13*d01, Vκ1-12*01,        Vκ1D-12*02, Vκ3-20*01, Vκ1-17*01, Vκ1D-39*01, Vκ3-11*01,        Vκ1D-16*01 and Vκ1-9*d01.    -   4. The cell or vertebrate of aspect 1-3 wherein the genome        comprises VH3-23*04, optionally wherein the heavy chain is        produced by recombination of a human J_(H) segment with a D        segment and said VH segment.    -   5. The cell or vertebrate of aspect 1-4 wherein the genome        comprises Vκ4-1*01, optionally wherein the heavy chain is        produced by recombination of a human JH segment with a D segment        and said VH segment    -   6. The cell or vertebrate of aspect 1-5 wherein the genome        comprises Vκ2-28*01, optionally wherein the heavy chain is        produced by recombination of a human JH segment with a D segment        and said VH segment

7 The cell or vertebrate of aspect 1-6 wherein the genome comprisesVκ1-12*01, optionally wherein the heavy chain is produced byrecombination of a human JH segment with a D segment and said VH segment

8 The cell or vertebrate of aspect 1-7 wherein the genome comprisesJH2*01 and Jκ2*01.

9 The cell or vertebrate of aspect 1-8 wherein the genome comprisesJH6*02 and Jκ4*01.

10 The cell or vertebrate of aspect 1-9 wherein said heavy chain locuscomprises VH3-23*04 recombined with JH2*01; or VH3-7*01 recombined withJH6*02.

11 The cell or vertebrate of aspect 1-10 wherein said light chain locuscomprises Vκ4-1*01 recombined with Jκ2*01; or Vκ2-28*01 recombined withJκ4*01.

12 The cell or vertebrate of any preceding aspect, wherein the genomecomprises VH3-23*04, JH2*01 (optionally recombined with the VH3-23*04),Vκ4-1*01 and Jκ2*01 (optionally recombined with the Vκ4-1*01).

13 The cell or vertebrate of any preceding aspect, wherein the genomecomprises VH3-7*01, J_(H)6*02 (optionally recombined with the VH3-7*01),Vκ2-28*01 and Jκ4*01 (optionally recombined with the Vκ2-28*01).

14 The cell or vertebrate of any preceding aspect, the heavy chain locusfurther comprising one or more additional heavy chain gene segments fromTable 7.

15 The cell or vertebrate of any preceding aspect, the heavy chain locuscomprising all of the gene segments of Table 1, all of the gene segmentsof Table 2, all of the gene segments of Table 3, all of the genesegments of Table 4, all of the gene segments of Table 5, all of thegene segments of Table 6 or all of the gene segments of Table 7.

16 The cell or vertebrate of any preceding aspect, the light chain locusfurther comprising one or more additional light chain segments fromTable 12.

17 The cell or vertebrate of any preceding aspect, the light chain locuscomprising all of the gene segments of Table 8, all of the gene segmentsof Table 9, all of the gene segments of Table 10, all of the genesegments of Table 11 or all of the gene segments of Table 12.

18 The cell or vertebrate of any preceding aspect, wherein the cell is ahybridoma or a B-cell (eg, an immortalised B cell).

19 The vertebrate or cell of any preceding aspect, wherein theimmunoglobulin heavy chains expressed by the mouse are essentiallyexclusively said heavy chains comprising human variable regions; andsaid heavy chains comprising human variable regions are expressed aspart of serum IgG1, IgG2b and IgM (and optionally IgG2a) antibodies.

20 A method of producing an antibody or an antigen binding fragmentthereof, the method comprising immunising a vertebrate of aspects 1 to19 with an antigen and recovering the antibody or fragment or recoveringa cell producing the antibody or fragment; optionally modifying theproduced antibody or fragment so that it comprises human constantregions,

wherein the variable domains of said antibody are encoded by

-   -   human gene segments JH2*01 or JH6*02 and one or more human VH        gene segments and one or more human D gene segments;    -   wherein the human VH gene segment is selected from the group        consisting of VH3-23*04, VH7-4-1*01, VH4-4*02, VH1-3*01,        VH3-13*01, VH3-7*01 and VH3-20*d01.    -   and    -   (b) human gene segments Jκ2*01 or Jκ4*01 and one or more human        Vκ gene segments,        wherein the human Vk gene segment is selected from the group        consisting of Vκ4-1*01, Vκ2-28*01, Vκ1D-13*d01, Vκ1-12*01,        Vκ1D-12*02, Vκ3-20*01, Vκ1-17*01, Vκ1D-39*01, Vκ3-11*01,        Vκ1D-16*01 and Vκ1-9*d01.

21 A method according to aspect 20 wherein the antigen is amulti-subunit human protein, a bacterial cytotoxin or a proteinexpressed as a transmembrane protein on human cells.

22 A method of producing an antibody or an antigen binding fragmentthereof, the method comprising isolating an antibody or antigen bindingfragment thereof from a cell according to any one of aspects 1 to 20;and optionally modifying the isolated antibody or fragment so that itcomprises human constant regions.

23 The method of aspect 20, further comprising

-   -   (a) isolating from the vertebrate a B-cell encoding an antibody        that binds the antigen,    -   (b) identifying or copying a nucleotide sequence of the B-cell        that encodes a VH domain of the antibody and/or identifying or        copying nucleotide sequence of the B-cell that encodes a VL        domain of the antibody; and    -   (c) using the sequence(s) to produce an isolated antibody or        fragment comprising the VH and/or VL domain; optionally wherein        the isolated antibody or fragment comprises human constant        regions.

24 The method of any one of aspects 20-23, wherein the antibody orfragment is produced by expression from a host cell selected from a CHO,HEK293, Cos or yeast (eg, Picchia) cell.

25 The method of aspect 24, wherein the antibody or fragment is producedby expression from a CHO cell.

26 The method of any one of aspects 20-25, further comprisingformulating the antibody or fragment with a diluent, carrier, excipientor a drug to produce a pharmaceutical composition for human medical use,optionally further comprising packaging the composition in a sterilecontainer, for example, a vial, tube, IV bag or syringe, furtheroptionally producing a kit comprising combining the package with a labelor instructions indicating use of the antibody composition for humanmedical use; optionally wherein the label or instructions comprises amedicament batch number and/or a marketing authorisation number, furtheroptionally an EMA or FDA marketing authorisation number.

27 The method of any one of aspects 20-26, wherein the vertebrate isaccording to any one of aspects 1 to 19 and the antibody or fragmentproduced by the method comprises

(a) a human heavy chain variable domain is a recombinant of human JH2*01or human JH6*02, one or more human VH gene segments and one or morehuman D gene segments; and(b) a human light chain variable domain is a recombinant of human JK2*01or human JK4*01 and one or more human Vκ gene segments.

28 The method of aspect 27, wherein the antibody or fragment produced bythe method comprises a human heavy chain variable domain recombinant ofVH3-23*04 and J_(H)2*01 and a human light chain variable domainrecombinant of Vκ4-1*01 and Jκ2*01.

29 The method of aspect 27, wherein the antibody or fragment produced bythe method comprises a human heavy chain variable domain recombinant ofVH3-7*01 and J_(H)6*02 and a human light chain variable domainrecombinant of Vκ2-28*01 and Jκ4*01.

30 An isolated antibody or fragment or kit produced by the method of anyone of aspects 20-29

31 An isolated antibody or antigen binding fragment thereof or kitproduced by the method of aspects 20-29 wherein the antibody ishumanised.

32 A composition comprising an antibody or fragment or kit produced bythe method of aspects 20-29 wherein the antibody or fragment is the onlytherapeutic agent.

33 A composition comprising an antibody or fragment or kit produced bythe method of aspects 20-29 further comprising an additional therapeuticagent, for example, an anti-hypercholesterolemia drug.

34 An isolated antibody or fragment or kit produced by the method of anyone of aspects 20-29 for human medical use.

35 Use of the isolated antibody or fragment produced by the method ofany one of aspects 20-29 in the manufacture of a medicament for humanmedical use.

36 The use of aspect 35, wherein the medicament is comprised by acomposition or kit as recited in aspect 26.

37 An isolated antibody or fragment or kit according to aspect 30-34, oruse according to aspect 35-36, which can specifically bind a humantarget selected from: proprotein convertase PC9, proprotein convertasesubtilisin kexin-9 (PCSK9), CD126, IL-4, IL-4 receptor, IL-6, IL-6receptor, IL-13, IL-18 receptor, Erbb3, cell ASIC1, ANG2, GDF-8,angiopoietin ligand-2, delta-like protein ligand 4, immunoglobulin G1,PDGF ligand, PDGF receptor or NGF receptor, toxin A or toxin B ofClostridium difficile, relaxin, CD48, Cd20, glucagon receptor, proteaseactivated receptor 2, TNF-Like ligand 1A (TL1A), angiopoietin related-2(AR-2), angiopoietin-like protein 4, RANKL, angiopoietin-like protein 3(ANGPTL3), delta-like ligand 4 (DLL4), big endothelin-1 (ET-1), activinA, receptor tyrosine kinases, for example human AR-1 and tyrosine kinasewith Ig and EGF homology domains (TIE) and TIE-2 receptor.

38 An isolated antibody or fragment or kit according to aspect 30-34, oruse according to aspect 35-36, for use in treatment of a human in needthereof, wherein the treatment comprises delivery of an effective amountof an antibody or fragment which specifically binds to a virus or anantigen selected from a human cytokine, growth factor, hormone, enzymeand a serum protein.

39 A host cell, for example a CHO, HEK293, Cos or yeast (eg, Picchia)cell, expressing an antibody or fragment produced by the method of anyone of aspects 20-29.

In the aspects described above, J_(H)6*02 can be replaced withJ_(H)6*01.

In a further aspect, the invention includes the following clauses:

CLAUSES

-   -   1. A non-human vertebrate cell (eg, a mouse cell or rat cell)        whose genome comprises human JH2*01 and/or human JH6*02, one or        more human VH gene segments and one or more human D gene        segments upstream of a constant region at an endogenous heavy        chain locus and human Jκ2*01 and/or human Jκ4*01 and one or more        human Vκ gene segments upstream of a constant region at an        endogenous light chain locus, wherein the gene segments in each        locus are operably linked to the constant region thereof so that        the cell is capable of producing an antibody heavy chain and an        antibody light chain, or where the cell can develop into a        vertebrate that expresses an antibody heavy chain and an        antibody light chain, wherein the heavy chain is produced by        recombination of the human JH2*01 and/or J_(H)6*02 segment with        a D segment and a VH segment and the light chain is produced by        recombination of the human Jκ2*01 and/or Jκ4*01 segment with a        Vκ segment.    -   2. A non-human vertebrate (eg, a mouse or rat) whose genome        comprises human JH2*01 and/or human JH6*02, one or more human VH        gene segments and one or more human D gene segments upstream of        a constant region at an endogenous heavy chain locus and human        Jκ2*01 and/or human Jκ4*01 and one or more human Vκ gene        segments upstream of a constant region at an endogenous light        chain locus, wherein the gene segments in each locus are        operably linked to the constant region thereof so that the        vertebrate is capable of producing an antibody heavy chain and        an antibody light chain, wherein the heavy chain is produced by        recombination of the human JH2*01 and/or JH6*02 segment with a D        segment and a VH segment and the antibody light chain is        produced by recombination of the human Jκ2*01 and/or Jκ4*01        segment with a Vκ segment.    -   3. The cell of clause 1 or vertebrate of clause 2, wherein the        genome comprises JH2*01 and Jκ2*01.    -   4. The cell of clause 1 or 3 or the vertebrate of clause 2 or 3,        wherein the genome comprises JH6*02 and Jκ4*01.    -   5. The cell or vertebrate of any preceding clause, wherein said        one or more human VH gene segments of the heavy chain locus        comprise or consist of one, more or all human VH gene segments        selected from the group consisting of VH3-23*04, VH7-4-1*01,        VH4-4*02, VH1-3*01, VH3-13*01, VH3-7*01 and VH3-20*d01.    -   6. The cell or vertebrate of clause 5, wherein said heavy chain        locus comprises VH3-23*04 recombined with JH2*01; or VH3-7*01        recombined with JH6*02.    -   7. The cell or vertebrate of any preceding clause, wherein said        one or more human Vκ gene segments comprise or consist of one,        more or all human VH gene segments selected from the group        consisting of Vκ4-1*01, Vκ2-28*01, Vκ1D-13*d01, Vκ1-12*01,        Vκ1D-12*02, Vκ3-20*01, Vκ1-17*01, Vκ1D-39*01, Vκ3-11*01,        Vκ1D-16*01 and Vκ1-9*d01.    -   8. The cell or vertebrate of clause 5, wherein said light chain        locus comprises Vκ4-1*01 recombined with Jκ2*01; or Vκ2-28*01        recombined with Jκ4*01.    -   9. The cell or vertebrate of any preceding clause, wherein the        genome comprises VH3-23*04, JH2*01 (optionally recombined with        the VH3-23*04), Vκ4-1*01 and Jκ2*01 (optionally recombined with        the Vκ4-1*01).    -   10. The cell or vertebrate of any preceding clause, wherein the        genome comprises VH3-7*01, JH6*02 (optionally recombined with        the VH3-7*01), Vκ2-28*01 and Jκ4*01 (optionally recombined with        the Vκ2-28*01).    -   11. The cell or vertebrate of any preceding clause, the heavy        chain locus further comprising one or more additional heavy        chain gene segments from Table 7.    -   12. The cell or vertebrate of any preceding clause, the heavy        chain locus comprising all of the gene segments of Table 1, all        of the gene segments of Table 2, all of the gene segments of        Table 3, all of the gene segments of Table 4, all of the gene        segments of Table 5, all of the gene segments of Table 6 or all        of the gene segments of Table 7.    -   13. The cell or vertebrate of any preceding clause, the light        chain locus further comprising one or more additional light        chain segments from Table 12.    -   14. The cell or vertebrate of any preceding clause, the light        chain locus comprising all of the gene segments of Table 8, all        of the gene segments of Table 9, all of the gene segments of        Table 10, all of the gene segments of Table 11 or all of the        gene segments of Table 12.    -   15. The cell or vertebrate of any preceding clause, wherein the        cell is a hybridoma or a B-cell (eg, an immortalised B cell).    -   16. The vertebrate or cell of any preceding clause, wherein the        immunoglobulin heavy chains expressed by the mouse are        essentially exclusively said heavy chains comprising human        variable regions; and said heavy chains comprising human        variable regions are expressed as part of serum IgG1, IgG2b and        IgM (and optionally IgG2a) antibodies.    -   17. A method of producing an antibody or an antigen binding        fragment thereof, the method comprising immunising a vertebrate        of clauses 2 to 16 with an antigen and recovering the antibody        or fragment or recovering a cell producing the antibody or        fragment; optionally modifying the produced antibody or fragment        so that it comprises human constant regions.    -   18. A method of producing an antibody or an antigen binding        fragment thereof, the method comprising isolating an antibody or        antigen binding fragment thereof from a cell according to any        one of clauses 1 and 3 to 16; and optionally modifying the        isolated antibody or fragment so that it comprises human        constant regions.    -   19. The method of clause 17 or clause 18, further comprising        -   (a) isolating from the vertebrate a B-cell encoding an            antibody that binds the antigen,        -   (b) identifying or copying a nucleotide sequence of the            B-cell that encodes a VH domain of the antibody and/or            identifying or copying nucleotide sequence of the B-cell            that encodes a VL domain of the antibody; and        -   (c) using the sequence(s) to produce an isolated antibody or            fragment comprising the VH and/or VL domain; optionally            wherein the isolated antibody or fragment comprises human            constant regions.    -   20. The method of any one of clauses 17-19, wherein the antibody        or fragment is produced by expression from a host cell selected        from a CHO, HEK293, Cos or yeast (eg, Picchia) cell.    -   21. The method of any one of clauses 17-20, further comprising        formulating the antibody or fragment with a diluent, carrier,        excipient or a drug to produce a pharmaceutical composition for        human medical use, optionally further comprising packaging the        composition in a sterile container, for example, a vial, tube,        IV bag or syringe, further optionally producing a kit comprising        combining the package with a label or instructions indicating        use of the antibody composition for human medical use;        optionally wherein the label or instructions comprises a        medicament batch number and/or a marketing authorisation number,        further optionally an EMA or FDA marketing authorisation number.    -   22. The method of any one of clauses 17-21, wherein the        vertebrate is according to any one of clauses 2 to 16 and the        antibody or fragment produced by the method comprises        -   (a) a human heavy chain variable domain is a recombinant of            human JH2*01 or human JH6*02, one or more human VH gene            segments and one or more human D gene segments; and        -   (b) a human light chain variable domain is a recombinant of            human Jκ2*01 or human Jκ4*01 and one or more human Vκ gene            segments.    -   23. The method of clause 22, wherein the antibody or fragment        produced by the method comprises a human heavy chain variable        domain recombinant of VH3-23*04 and JH2*01 and a human light        chain variable domain recombinant of Vκ4-1*01 and Jκ2*01.    -   24. The method of clause 22, wherein the antibody or fragment        produced by the method comprises a human heavy chain variable        domain recombinant of VH3-7*01 and JH6*02 and a human light        chain variable domain recombinant of Vκ2-28*01 and Jκ4*01.    -   25. An isolated antibody or antigen binding fragment thereof        obtained or obtainable by the method of any one of clauses        17-24, wherein a heavy and/or light chain variable domain of the        antibody or fragment comprises one or more mouse or rat        activation-induced deaminase (AID) pattern somatic mutations        and/or mouse or rat terminal deoxynucleotidyl transferase (TdT)        pattern junctional mutations.    -   26. An isolated antibody or antigen binding fragment thereof        obtained or obtainable by the method of any one of clauses        17-24, or an antibody according to clause 25 comprising a human        heavy chain Fc fragment, optionally that binds a gamma receptor,        and further optionally a human constant light chain.    -   27. An isolated antibody or antigen binding fragment thereof        obtained or obtainable by the method of any one of clauses        17-24, or an antibody according to clause 25 or clause 26        comprising an antigen binding site capable of specifically        binding to a virus or an antigen selected from a human cytokine,        growth factor, hormone, enzyme and a serum protein.    -   28. An isolated antibody or antigen binding fragment thereof        obtained or obtainable by the method of any one of clauses        17-24, or an antibody according to any one of clauses 25-27,        wherein said antibody or fragment is glycosylated, optionally        comprising CHO, HEK293, Cos or yeast (eg, Picchia) cell        glycosylation.    -   29. An isolated antibody or antigen binding fragment thereof,        wherein the variable domains of said antibody are encoded by        -   (a) human gene segments JH2*01 or JH6*02 and one or more            human VH gene segments and one or more human D gene            segments; and        -   (b) human gene segments Jκ2*01 or Jκ4*01 and one or more            human Vκ gene segments.    -   30. The antibody of clause 29, wherein the variable domains are        encoded by the human gene segments (i) JH2*01 and VH3-23*04 for        the VH domain and (ii) Vκ4-1*01 and Jκ2*01 for the VL domain.    -   31. The antibody of clause 29, wherein the variable domains are        encoded by the human gene segments (i) JH6*02 and VH3-7*01 for        the VH domain and (ii) Vκ2-28*01 and Jκ4*01 for the VL domain.    -   32. An isolated antibody or antigen binding fragment thereof of        any one of clauses 25-31, wherein the antibody is humanised.    -   33. A composition comprising an antibody or fragment of any one        of clauses 25-32, wherein the antibody or fragment is the only        therapeutic agent.    -   34. A composition comprising an antibody or fragment of any one        of clauses 25-32, further comprising an additional therapeutic        agent, for example, an anti-hypercholesterolemia drug.    -   35. The isolated antibody or fragment produced by the method of        any one of clauses 17-24 or the isolated antibody or fragment of        any one of clauses 25 to 34 for human medical use.    -   36. Use of the isolated antibody or fragment produced by the        method of any one of clauses 17-24 or the isolated antibody or        fragment of any one of clauses 25-34 in the manufacture of a        medicament for human medical use.    -   37. The use of clause 36, wherein the medicament is comprised by        a composition or kit as recited in clause 21.    -   38. A method of medical treatment comprising delivery an        effective amount of isolated antibody or fragment thereof        according to clauses 25-34 to a human in need thereof.

-   39. A host cell, for example a CHO, HEK293, Cos or yeast (eg,    Picchia) cell, expressing an antibody or fragment as defined in any    one of clauses 25-34.

In the clauses described above, JH6*02 can be replaced with JH6*01.

In a further aspect, the invention includes the following provisions:

-   -   1. A non-human vertebrate (eg, a mouse or rat) cell whose genome        comprises human VH, D and JH gene segments upstream of a        constant region at an endogenous heavy chain locus and human VL        and JL gene segments upstream of a constant region at an        endogenous light chain locus, wherein the gene segments are        operably linked to the constant region thereof so that the cell        is capable of expressing immunoglobulin heavy and light chains        comprising human VH and VL domains respectively, wherein the        heavy chain locus comprises a human 01 allele VH gene segment        capable of recombining with a human D and JH gene segment to        produce a VH domain, wherein the light chain locus comprises a        human 01 allele VL gene segment capable of recombining with a        human JL gene segment to produce a VL domain, or wherein the        cell can develop into a vertebrate that expresses said VH and VL        domains.    -   2. A non-human vertebrate (eg, a mouse or rat) whose genome        comprises human VH, D and J_(H) gene segments upstream of a        constant region at an endogenous heavy chain locus and human VL        and JL gene segments upstream of a constant region at an        endogenous light chain locus, wherein the gene segments are        operably linked to the constant region thereof so that the        vertebrate is capable of expressing immunoglobulin heavy and        light chains comprising human VH and VL domains respectively,        wherein the heavy chain locus comprises a human 01 allele VH        gene segment capable of recombining with a human D and JH gene        segment to produce a VH domain and wherein the light chain locus        comprises a human 01 allele VL gene segment capable of        recombining with a human JL gene segment to produce a VL domain.    -   3. The cell of provision 1 or vertebrate of provision 2, wherein        one or both 01 alleles is a d01 allele.    -   4. The cell or vertebrate of any preceding provision, wherein        the VH and VL domains form an antigen binding site.    -   5. The cell or vertebrate of any preceding provision, wherein        the heavy chain locus comprises no second human allele of said        VH gene segment.    -   6. The cell or vertebrate of any preceding provision, wherein        the light chain locus comprises no second human allele of said        VL gene segment.    -   7. The cell or vertebrate of any preceding provision, wherein        the heavy chain locus comprises a human 01 allele D gene segment        capable of recombining with a human JH gene segment and said        human VH segment to produce said VH domain;        -   optionally wherein the heavy chain locus comprises no second            human allele of said D gene segment.    -   8. The cell or vertebrate of any preceding provision, wherein        the heavy chain locus comprises a human 02 allele JH gene        segment (eg, JH6*02) capable of recombining with a human D gene        segment and said human VH segment to produce said VH domain;        optionally wherein the heavy chain locus comprises no second        human allele of said JH gene segment.    -   9. The cell or vertebrate of any preceding provision, wherein        the light chain locus comprises a human 01 allele JL gene        segment capable of recombining said human VL segment to produce        said VL domain; optionally wherein the light chain locus        comprises no second human allele of said JL gene segment.    -   10. The cell or vertebrate of any preceding provision, wherein        the genome comprises VH3-23*04, JH2*01 (optionally recombined        with the VH3-23*04), Vκ4-1*01 and Jκ2*01 (optionally recombined        with the Vκ4-1*01).    -   11. The cell or vertebrate of any preceding provision, wherein        the genome comprises VH3-7*01, J_(H)6*02 (optionally recombined        with the VH3-7*01), Vκ2-28*01 and Jκ4*01 (optionally recombined        with the Vκ2-28*01).    -   12. The cell or vertebrate of any preceding provision, wherein        said light chain locus comprises or consists of one, more or all        human VL gene segments selected from the group consisting of        Vκ4-1*01, Vκ2-28*01, Vκ1D-13*d01, Vκ1-12*01, Vκ1D-12*02,        Vκ3-20*01, Vκ1-17*01, Vκ1D-39*01, Vκ3-11*01, Vκ1D-16*01 and        Vκ1-9*d01.    -   13. The cell or vertebrate of any one of provisions 1 to 9,        wherein said light chain locus comprises or consists of one,        more or all human VL gene segments selected from the group        consisting of Jκ4*01 and Jκ2*01.    -   14. The cell or vertebrate of any preceding provision, wherein        said heavy chain locus comprises or consists of one, more or all        human VH gene segments selected from the group consisting of        VH3-23*04, VH7-4-1*01, VH4-4*02, VH1-3*01, VH3-13*01, VH3-7*01        and VH3-20*d01.    -   15. The cell or vertebrate of any one of provisions 1 to 9 or        14, wherein the light chain locus comprises Jλ2*01.    -   16. The cell or vertebrate of any one of provisions 1 to 6,        wherein said VH domain is a recombinant of human VH, D and JH        segments, wherein the VH is selected from the group consisting        of one, more or all 01 allele gene VH segments of Table 7 and/or        the VL domain is a recombinant of (i) human Vκ and Jκ gene        segments, wherein the Vκ is selected from the group consisting        of one, more or all 01 allele Vκ gene segments of Table 12        or (ii) human Vλ and Jλ gene segments, the Vλ being selected        from the group consisting of one, more or all 01 allele Vλ gene        segments of Table 18; optionally wherein the VH and/or VL        domains are expressed as IgG antibodies.    -   17. The cell or vertebrate of any one of provisions 1 to 6 and        16, wherein said VH domain is a recombinant of the human VH, D        and J_(H) segments of Table 1, Table 2, Table 3, Table 4, Table        5, Table 6 or Table 7 and/or the VL domain is a recombinant        of (i) the human Vκ and Jκ gene segments of Table 8, Table 9,        Table 10, Table 11 or Table 12; or (ii) the human Vλ and Jλ gene        segments of Table 13, Table 14, Table 15, Table 16, Table 17 or        Table 18.    -   18. The vertebrate of any one of provisions 2 to 17, wherein (a)        the heavy chain locus comprises the human VH, D and JH segments        of a Table selected from Table 1, Table 2, Table 3, Table 4,        Table 5, Table 6 and Table 7 and/or (b) the light chain locus        comprises (i) the human Vκ and Jκ gene segments of a Table        selected from Table 8, Table 9, Table 10, Table 11 and Table 12;        or (ii) the human Vλ and Jλ gene segments of a Table selected        from Table 13, Table 14, Table 15, Table 16, Table 17 and Table        18; and wherein the vertebrate expresses one or a plurality of        VH domains each being a recombinant of human VH, D and J_(H)        segments from a selected Table of (a) and/or expresses one or a        plurality of VL domains each being a recombinant of human VL and        JL segments from a selected Table of (b); optionally wherein        such VH domains and VL domains form antigen binding sites.    -   19. A non-human vertebrate (eg, a mouse or rat) whose genome        comprises human VH, D and JH gene segments upstream of a        constant region at an endogenous heavy chain locus and human VL        and JL gene segments upstream of a constant region at an        endogenous light chain locus, wherein the gene segments are        operably linked to the constant region thereof so that the        vertebrate is capable of expressing immunoglobulin heavy and        light chains comprising human VH and VL domains respectively,        wherein (a) the heavy chain locus comprises the human VH, D and        JH segments of a Table selected from Table 1, Table 2, Table 3,        Table 4, Table 5, Table 6 and Table 7 and/or (b) the light chain        locus comprises (i) the human Vκ and Jκ gene segments of a Table        selected from Table 8, Table 9, Table 10, Table 11 and Table 12;        or (ii) the human Vλ and Jλ gene segments of a Table selected        from Table 13, Table 14, Table 15, Table 16, Table 17 and Table        18; and wherein the vertebrate expresses one or a plurality of        VH domains each being a recombinant of human VH, D and J_(H)        segments from a selected Table of (a) and/or expresses one or a        plurality of VL domains each being a recombinant of human VL and        JL segments from a selected Table of (b); optionally wherein        such VH domains and VL domains form antigen binding sites.    -   20. The cell of any preceding provision, wherein said cell is an        ES cell, a hybridoma cell or an immortalised B cell.    -   21. The vertebrate of any preceding provision, wherein said        vertebrate is comprised within a container having an air filter.    -   22. A population of at least 90 cells, wherein said cells are        according to any one of provisions 1 to 17, 19 or 20.    -   23. The cell, vertebrate or population of any preceding        provision, wherein said light chain locus constant region is a        kappa or lambda constant region; optionally a mouse or rat        constant region.    -   24. The cell, vertebrate or population of any preceding        provision, wherein the heavy chain locus is a mouse or rat        constant region (eg, comprising a gamma constant gene segment).    -   25. The cell, vertebrate or population of any preceding        provision, wherein said endogenous light chain locus is a kappa        or lambda locus; optionally wherein the genome comprises at        least the V and J gene segments of Table 8 at an endogenous        light chain locus and/or at least the V and J gene segments of        Table 13 at an endogenous light chain locus.    -   26. The cell, vertebrate or population of any one of provisions        1 to 9 or 14 to 23, wherein said light chains comprise        immunoglobulin light chains comprising human variable regions        that derived from recombination of human Vλ and Jλ gene segments        selected from the group consisting of Vλ and Jλ gene segments of        Table 18, optionally each such variable region being expressed        with a constant region encoded by a Cλ gene segment selected        from the group consisting of the Cλ gene segments of Table 18.    -   27. The cell, vertebrate or population of any preceding        provision, wherein the vertebrate is a mouse C57Bl/6J, 129S5 or        129Sv strain or a cross between C57Bl/6J and a 129S5 or 129Sv        strain.    -   28. The cell, vertebrate or population of any preceding        provision, wherein the immunoglobulin heavy chains expressed by        the cell or vertebrate are essentially exclusively said heavy        chains comprising human variable regions; and said heavy chains        comprising human variable regions are expressed as part of serum        IgG1, IgG2b and IgM (and optionally IgG2a) antibodies.    -   29. The cell, vertebrate or population of any preceding        provision, wherein the vertebrate expresses light chains        comprising human lambda variable regions and at least 60%, 70%        or 80% of the variable regions of such light chains are derived        from recombination of human Vλ and Jλ gene segments.    -   30. A non-human vertebrate (eg, a mouse or rat) or cell whose        genome comprises an Ig gene segment repertoire produced by        targeted insertion of human Ig gene segments into one or more        endogenous Ig loci, the genome comprising human Vλ and Jλ gene        segments upstream of a constant region, wherein the human Vλ and        Jλ gene segments have been provided by insertion into an        endogenous light chain locus of the vertebrate or cell, wherein        the vertebrate comprises immunoglobulin light chains comprising        lambda variable regions (lambda light chains) or the cell can        develop into a vertebrate that expresses said immunoglobulin        light chains, wherein the lambda light chains comprise        immunoglobulin light chains comprising lambda variable regions        derived from recombination of human Vλ and Jλ gene segments;        wherein at least 80% of the variable regions of the lambda light        chains expressed by the vertebrate are human Vλ and Jλ gene        segment recombinants; wherein the vertebrate or cell is        according to any one of provisions 1 to 9 or 14 to 29.    -   31. A non-human vertebrate (eg, a mouse or rat) or cell whose        genome comprises an Ig gene segment repertoire produced by        targeted insertion of human Ig gene segments into one or more        endogenous Ig loci, the genome comprising human Vλ and Jλ gene        segments upstream of a constant region, wherein the human Vλ and        Jλ gene segments are selected from one, more or all of the        segments of Table 18 and have been provided by insertion into an        endogenous light chain locus of the vertebrate or cell, wherein        the vertebrate comprises immunoglobulin light chains comprising        lambda variable regions (lambda light chains) or the cell can        develop into a vertebrate that expresses said immunoglobulin        light chains, wherein the lambda light chains comprise        immunoglobulin light chains comprising lambda variable region        recombinants of one or a plurality of human Vλ and Jλ gene        segment pairs, wherein each gene segment is selected from the        human Vλ and Jλ gene segments of Table 18.    -   32. A method of producing an antibody or an antigen binding        fragment thereof, the method comprising immunising a vertebrate        of any one of provisions 2 to 19, 21 or 23 to 31 with an antigen        and recovering the antibody or fragment or recovering a cell        producing the antibody or fragment; optionally modifying the        produced antibody or fragment so that it comprises human        constant regions.    -   33. A method of producing an antibody or an antigen binding        fragment thereof, the method comprising isolating an antibody or        antigen binding fragment thereof from a cell according to any        one of provisions 1, 3 to 17, 19 and 23 to 31; and optionally        modifying the isolated antibody or fragment so that it comprises        human constant regions.    -   34. The method of provision 33, further comprising        -   (a) isolating from the vertebrate a B-cell encoding an            antibody that binds the antigen,        -   (b) identifying or copying a nucleotide sequence of the            B-cell that encodes a VH domain of the antibody and/or            identifying or copying nucleotide sequence of the B-cell            that encodes a VL domain of the antibody; and        -   (c) using the sequence(s) to produce an isolated antibody or            fragment comprising the VH and/or VL domain; optionally            wherein the isolated antibody or fragment comprises human            constant regions.    -   35. The method of any one of provisions 32 to 34, wherein the        antibody or fragment is produced by expression from a host cell        selected from a CHO, HEK293, Cos or yeast (eg, Picchia) cell.    -   36. The method of provision any one of provisions 32 to 35,        further comprising formulating the antibody or fragment with a        diluent, carrier, excipient or a drug to produce a        pharmaceutical composition for human medical use, optionally        further comprising packaging the composition in a sterile        container, for example, a vial, tube, IV bag or syringe, further        optionally producing a kit comprising combining the package with        a label or instructions indicating use of the antibody        composition for human medical use; optionally wherein the label        or instructions comprises a medicament batch number and/or a        marketing authorisation number, further optionally an EMA or FDA        marketing authorisation number.    -   37. The method of any one of provisions 32 to 35, wherein the        vertebrate is according to any preceding provision and the        antibody or fragment produced by the method comprises        -   (a) a human heavy chain variable domain recombinant of human            VH, D and J_(H) gene segments selected from the group            consisting of VH, D and J_(H) gene segments of Table 7; and        -   (b) a human light chain variable domain recombinant of human            V and J gene segments selected from the V and J gene            segments of Table 12 or selected from the V and J gene            segments of Table 18.    -   38. An isolated antibody or antigen binding fragment thereof        obtained or obtainable by the method of any one of provisions 32        to 35, wherein a heavy and/or light chain variable domain of the        antibody or fragment comprises one or more mouse or rat        activation-induced deaminase (AID) pattern somatic mutations        and/or mouse or rat terminal deoxynucleotidyl transferase (TdT)        pattern junctional mutations.    -   39. An isolated antibody or antigen binding fragment thereof        obtained or obtainable by the method of provision 32 to 35, or        an antibody according to provision 38 comprising a human heavy        chain Fc fragment, optionally that binds a gamma receptor, and        further optionally a human constant light chain.    -   40. An isolated antibody or antigen binding fragment thereof        obtained or obtainable by the method of any one of provisions 32        to 35, or an antibody according to provision 38 or provision 39        comprising an antigen binding site capable of specifically        binding to a virus or an antigen selected from a human cytokine,        growth factor, hormone, enzyme and a serum protein.    -   41. An isolated antibody or antigen binding fragment thereof        obtained or obtainable by the method of any one of provisions 32        to 35, or an antibody according to any one of provisions 38 to        40, wherein said antibody or fragment is glycosylated,        optionally comprising CHO, HEK293, Cos or yeast (eg, Picchia)        cell glycosylation.    -   42. An isolated antibody or antigen binding fragment thereof,        wherein the variable domains of said antibody are encoded by (i)        human VH, D and J_(H) segments selected from the group        consisting of any gene segment of Table 7 and/or (i) human Vκ        and Jκ gene segments selected from the group consisting of any        Vκ and Jκ gene segments of Table 12 or (ii) human Vλ and Jλ gene        segments selected from the group consisting of any Vλ and Jλ        gene segments of Table 18.    -   43. An isolated antibody or antigen binding fragment thereof of        any one of provisions 38 to 42, wherein the antibody or fragment        specifically binds to a virus or an antigen selected from a        human cytokine, growth factor, hormone, enzyme and a serum        protein.    -   44. An isolated antibody or antigen binding fragment thereof of        any one of provisions 38 to 43, wherein the antibody is        humanised.    -   45. A composition comprising an antibody or fragment of any one        of provisions 38 to 44, wherein the antibody or fragment is the        only therapeutic agent.    -   46. A composition comprising an antibody or fragment of any one        of provisions 38 to 44, further comprising an additional        therapeutic agent, for example, an anti-rheumatic drug, such as        a disease modifying anti-rheumatic drug, or an anti-cancer drug        or an anti-hypercholesterolaemia drug.    -   47. The isolated antibody or fragment produced by the method of        any one of provisions 32 to 35 or the isolated antibody or        fragment of any one of provisions 38 to 46 for human medical        use.    -   48. Use of the isolated antibody or fragment produced by the        method of any one of provisions 32 to 35 or the isolated        antibody or fragment of provision 38 to 46 in the manufacture of        a medicament for human medical use.    -   49. The use of provision 48, wherein the medicament is comprised        by a composition or kit as recited in provision 36.    -   50. A method of medical treatment comprising delivery an        effective amount of isolated antibody or fragment thereof        according to provisions 38 to 46 to a human in need thereof.    -   51. A host cell, for example a CHO, HEK293, Cos or yeast (eg,        Picchia) cell, expressing an antibody or fragment as defined in        any one of provisions 38 to 43.

In the provisions described above, JH6*02 can be replaced with JH6*01.

The Following Definitions Apply to any Configuration, Aspect, Clause,Provision, Example or Embodiment of the Invention.

“Derived from” is used in the ordinary sense of the term. Exemplarysynonyms include “produced as”, “resulting from”, “received from”,“obtained from”, “a product of”, “consequence of”, and “modified from”For example, a human variable region of a heavy chain can be derivedfrom recombination of human VH, D and J_(H) gene segments and thisreflects the in vivo recombination of these gene segments in, forexample, a transgenic heavy chain locus according to the invention withany accompanying mutation (eg, junctional mutation).

Samples from which B-cells can be obtained include but are not limitedto blood, serum, spleen, splenic tissue, bone marrow, lymph, lymph node,thymus, and appendix. Antibodies and immunoglobulin chains can beobtained from each of the previous-mentioned samples and also from thefollowing non-limiting list of B-cells, ascites fluid, hybridomas, andcell cultures.

“Plurality” is used in the ordinary sense of the term and means “atleast one” or “more than one”.

The term “germline configuration” refers to a germline genomicconfiguration. For example, human immunoglobulin gene segments of atransgenic immunoglobulin locus are in a germline configuration when therelative order of the gene segments is the same as the order ofcorresponding gene segments in a human germline genome. For example,when the transgenic locus is a heavy chain locus of the inventioncomprising hypothetical human immunoglobulin gene segments A, B and C,these would be provided in this order (5′ to 3′ in the locus) when thecorresponding gene segments of a human germline genome comprises thearrangement 5′-A-B-C-3′. In an example, when elements of a humanimmunoglobulin locus (eg, gene segments, enhancers or other regulatoryelements) are provided in a transgenic immunoglobulin locus according tothe invention, the human Ig locus elements are in germline configurationwhen when the relative order of the gene segments is the same as theorder of corresponding gene segments in a human germline genome andhuman sequences between the elements are included, these correspondingto such sequences between corresponding elements in the human germlinegenome. Thus, in a hypothetical example the transgenic locus compriseshuman elements in the arrangement 5′-A-S1-B-S2-C-S3-3′, wherein A, B andC are human immunoglobulin gene segments and S1-S3 are human inter-genesegment sequences, wherein the corresponding arrangement5′-A-S1-B-S2-C-S3-3′ is present in a human germline genome. For example,this can be achieved by providing in a transgenic immunoglobulin locusof the invention a DNA insert corresponding to the DNA sequence from Ato C in a human germline genome (or the insert comprising the DNAsequence from A to C). The arrangements in human germline genomes andimmunoglobulin loci are known in the art (eg, see the IMGT at the WorldWide Web (see above), Kabat and other antibody resources referencedherein).

The term “antibody” includes monoclonal antibodies (including fulllength antibodies which have an immunoglobulin Fc region), antibodycompositions with polyepitopic specificity, multispecific antibodies(e.g., bispecific antibodies, diabodies, and single-chain molecules, aswell as antibody fragments (e.g., dAb, Fab, F(ab′)2, and Fv). The term“antibody” also includes H2 antibodies that comprise a dimer of a heavychain (5′-VH-(optional Hinge)-CH2-CH3-3′) and are devoid of a lightchain (akin to naturalluy-occurring H2 antibodies; see, eg, Nature. 1993Jun. 3; 363(6428):446-8; Naturally occurring antibodies devoid of lightchains; Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G,Hamers C, Songa E B, Bendahman N, Hamers R). Thus, in an embodiment ofthe present invention, RNA produced from the transgenic heavy chainlocus encodes for heavy chains that re devoid of a CH1 gene segment andcomprise no functional antibody light chain. In an example, RNA producedfrom the transgenic heavy chain locus encodes for VH single variabledomains (dAbs; domain antibodies). These can optionally comprise aconstant region.

The term “immunoglobulin” (Ig) is used interchangeably with “antibody”herein.

An “isolated” antibody is one that has been identified, separated and/orrecovered from a component of its production environment (e.g.,naturally or recombinantly). Preferably, the isolated polypeptide isfree of association with all other components from its productionenvironment, eg, so that the antibody has been isolated to anFDA-approvable or approved standard. Contaminant components of itsproduction environment, such as that resulting from recombinanttransfected cells, are materials that would typically interfere withresearch, diagnostic or therapeutic uses for the antibody, and mayinclude enzymes, hormones, and other proteinaceous or non-proteinaceoussolutes. In preferred embodiments, the polypeptide will be purified: (1)to greater than 95% by weight of antibody as determined by, for example,the Lowry method, and in some embodiments, to greater than 99% byweight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under non-reducing orreducing conditions using Coomassie blue or, preferably, silver stain.Isolated antibody includes the antibody in situ within recombinant cellssince at least one component of the antibody's natural environment willnot be present. Ordinarily, however, an isolated polypeptide or antibodywill be prepared by at least one purification step.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include dAb, Fab, Fab′, F(ab′)2and Fv fragments; diabodies; linear antibodies; single-chain antibodymolecules and multispecific antibodies formed from antibody fragments.

An antibody that “specifically binds to” or is “specific for” aparticular polypeptide, antigen, or epitope is one that binds to thatparticular polypeptide, antigen, or epitope without substantiallybinding to other polypeptides, antigens or epitopes. For example,binding to the antigen or epitope is specific when the antibody bindswith a K_(D) of 100 μM or less, 10 μM or less, 1 μM or less, 100 nM orless, eg, 10 nM or less, 1 nM or less, 500 μM or less, 100 μM or less,or 10 μM or less. The binding affinity (K_(D)) can be determined usingstandard procedures as will be known by the skilled person, eg, bindingin ELISA and/or affinity determination using surface plasmon resonance(eg, Biacore™ or KinExA™ solution phase affinity measurement which candetect down to fM affinities (Sapidyne Instruments, Idaho)).

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the USA Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, including humans. A “pharmaceutically acceptable carrier,excipient, or adjuvant” refers to a carrier, excipient, or adjuvant thatcan be administered to a subject, together with an agent, e.g., anyantibody or antibody chain described herein, and which does not destroythe pharmacological activity thereof and is nontoxic when administeredin doses sufficient to deliver a therapeutic amount of the agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1, part 1 illustrates the first and second BACs used for insertioninto mouse endogenous light chain loci. The human DNA in each BAC isshown. Part 2 of FIG. 1 shows the insertion point of human lambda Iglocus DNA into the mouse endogenous kappa chain locus. Part 3 of FIG. 1shows the insertion point of human lambda Ig locus DNA into the mouseendogenous lambda chain locus.

FIG. 2 shows the results of FACS analysis to determine mouse and humanCλ expression (and thus correspondingly mouse and human variable regionexpression) in B220⁺ splenic B cells from P1 homozygous mice (P1/P1)compared to wild-type mice (WT).

FIG. 3A shows the results of FACS analysis to determine mouse Cκ and Cλexpression in B220⁺ splenic B cells from P2 homozygous mice (P2/P2)compared to wild-type mice (WT). No detectable mouse Cκ expression wasseen.

FIG. 3B shows the results of FACS analysis to determine human Cλexpression (and thus correspondingly human variable region expression)in B220⁺ splenic B cells from P2 homozygous mice (P2/P2) compared towild-type mice (WT).

FIG. 4 shows human Vλ usage in P2 homozygous mice (P2/P2) and typical Vλusage in humans (inset).

FIG. 5 shows human Jλ usage in P2 homozygous mice (P2/P2) and typical Jλusage in humans (inset).

FIG. 6 shows Vλ usage is very high in P2 homozygous mice (P2/P2).

FIG. 7 shows the distribution of mouse Vκ and human Vλ gene segmentusage from the chimaeric kappa locus in P2 homozygous mice (P2/P2).

FIG. 8 illustrates RSS arrangement in the lambda and kappa loci.

FIG. 9A shows the results of FACS analysis to determine mouse and humanCλ expression (and thus correspondingly mouse and human variable regionexpression) in B220⁺ splenic B cells from L2 homozygous mice in whichendogenous kappa chain expression has been inactivated (L2/L2; KA/KA)compared to mice having no human lambda DNA inserted and in whichendogenous kappa chain expression has been inactivated (KA/KA). Veryhigh human Vλ usage was seen in the L2/L2; KA/KA) mice, almost to theexclusion of mouse Vλ use.

FIG. 9B: Splenic B-Cell Compartment Analysis. This figure shows theresults of FACS analysis on splenic B-cells from transgenic L2/L2; KA/KAmice (L2 homozygotes; homozygous for human lambda gene segment insertioninto endogenous lambda loci; endogenous kappa chain expression havingbeen inactivated) compared with splenic B-cells from mice expressingonly mouse antibodies (KA/KA mice). The results show that the splenicB-cell compartments in the mice of the invention are normal (ie,equivalent to the compartments of mice expressing only mouse antibodychains).

FIG. 10: B-cell development and markers in the bone marrow and spleniccompartments.

FIG. 11A: Splenic B-Cell Compartment Analysis. This figure shows theresults of FACS analysis on splenic B-cells from transgenic S1F/HA, KA/+mice of the invention expressing heavy chain variable regions which areall human (where endogenous heavy chain expression has been inactivatedby inversion), compared with splenic B-cells from mice expressing onlymouse antibodies. The results show that the splenic B-cell compartmentsin the mice of the invention are normal (ie, equivalent to thecompartments of mice expressing only mouse antibody chains).

S1F/HA, +/KA=(i) S1F—first endogenous heavy chain allele has one humanheavy chain locus DNA insertion, endogenous mouse VDJ region has beeninactivated by inversion and movement upstream on the chromosome; (ii)HA—second endogenous heavy chain allele has been inactivated (byinsertion of an endogenous interrupting sequence); (iii)+—firstendogenous kappa allele is a wild-type kappa allele; and (iv) KA—thesecond endogenous kappa allele has been inactivated (by insertion of anendogenous interrupting sequence). This arrangement encodes exclusivelyfor heavy chains from the first endogenous heavy chain allele.

FIG. 11B: Splenic B-Cell Compartment Analysis. This figure shows theresults of FACS analysis on splenic B-cells from transgenic S1F/HA,K2/KA mice of the invention expressing heavy chain variable regionswhich are all human (where endogenous heavy chain expression has beeninactivated by inversion) and human kappa chain variable regions,compared with splenic B-cells from +/HA, K2/KA mice. The results showthat the splenic B-cell compartments in the mice of the invention arenormal.

S1F/HA, K2/KA=(i) K2—the first endogenous kappa allele has two kappachain locus DNA insertions between the most 3′ endogenous Jκ and themouse Cκ, providing an insertion of 14 human Vκ and Jκ1-Jκ5; and (ii)KA—the second endogenous kappa allele has been inactivated (by insertionof an endogenous interrupting sequence). This arrangement encodesexclusively for heavy chains comprising human variable regions andsubstantially kappa light chains from the first endogenous kappa allele.

+/HA, K2/KA—this arrangement encodes for mouse heavy chains and humankappa chains.

FIG. 12A: Bone marrow B progenitor compartment analysis. This figureshows the results of FACS analysis on bone marrow (BM) B-cells fromtransgenic S1F/HA, KA/+ mice of the invention expressing heavy chainvariable regions which are all human (where endogenous heavy chainexpression has been inactivated by inversion), compared with BM B-cellsfrom mice expressing only mouse antibodies. The results show that the BMB-cell compartments in the mice of the invention are normal (ie,equivalent to the compartments of mice expressing only mouse antibodychains).

FIG. 12B: Bone marrow B progenitor compartment analysis. This figureshows the results of FACS analysis on bone marrow (BM) B-cells fromtransgenic S1F/HA, K2/KA mice of the invention expressing heavy chainvariable regions which are all human (where endogenous heavy chainexpression has been inactivated by inversion) and human kappa chainvariable regions, compared with BM B-cells from +/HA, K2/KA mice. Theresults show that the BM B-cell compartments in the mice of theinvention are normal.

FIG. 13: shows Ig quantification for subtype and total Ig in variousmice: S1F/HA, KA/+=(i) S1F—first endogenous heavy chain allele has onehuman heavy chain locus DNA insertion, endogenous mouse VDJ region hasbeen inactivated by inversion and movement upstream on the chromosome;(ii) HA—second endogenous heavy chain allele has been inactivated (byinsertion of an endogenous interrupting sequence); (iii) KA—the firstendogenous kappa allele has been inactivated (by insertion of anendogenous interrupting sequence); and (iv)+—second endogenous kappaallele is a wild-type kappa allele. This arrangement encodes exclusivelyfor heavy chains from the first endogenous heavy chain allele.

S1F/HA, K2/KA=(i) K2—the first endogenous kappa allele has two kappachain locus DNA insertions between the most 3′ endogenous Jκ and themouse Cκ, providing an insertion of 14 human Vκ and Jκ1-Jκ5; and (ii)KA—the second endogenous kappa allele has been inactivated (by insertionof an endogenous interrupting sequence). This arrangement encodesexclusively for heavy chains comprising human variable regions andsubstantially kappa light chains from the first endogenous kappa allele.

+/HA, K2/+—this arrangement encodes for mouse heavy chains and bothmouse and human kappa chains.

+/HA, +/KA—this arrangement encodes for mouse heavy and kappa chains.

In this figure, “Sum Ig” is the sum of IgG and IgM isotypes.

FIG. 14: shows Ig quantification for subtype and total Ig in variousmice: S1F/HA, K2/KA (n=15) and 12 mice expressing only mouse antibodychains (+/HA, +/KA (n=6) and wild-type mice (WT; n=6)).

DETAILED DESCRIPTION OF THE INVENTION

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine study, numerous equivalents to the specific proceduresdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the claims.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof is intended to include atleast one of: A, B, C, AB, AC, BC, or ABC, and if order is important ina particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As a source of antibody gene segment sequences, the skilled person willalso be aware of the following available databases and resources(including updates thereof) the contents of which are incorporatedherein by reference:

The Kabat Database (G. Johnson and T. T. Wu, 2002; World Wide Web (www)kabatdatabase.com). Created by E. A. Kabat and T. T. Wu in 1966, theKabat database publishes aligned sequences of antibodies, T-cellreceptors, major histocompatibility complex (MHC) class I and IImolecules, and other proteins of immunological interest. A searchableinterface is provided by the SeqhuntII

tool, and a range of utilities is available for sequence alignment,sequence subgroup classification, and the generation of variabilityplots. See also Kabat, E. A., Wu, T. T., Perry, H., Gottesman, K., andFoeller, C. (1991) Sequences of Proteins of Immunological Interest, 5thed., NIH Publication No. 91-3242, Bethesda, Md., which is incorporatedherein by reference, in particular with reference to human gene segmentsfor use in the present invention.

KabatMan (A. C. R. Martin, 2002; World Wide Web (www)bioinf.org.uk/abs/simkab.html). This is a web interface to make simplequeries to the Kabat sequence database.

IMGT (the International ImMunoGeneTics Information System®; M.-P.Lefranc, 2002; World Wide Web (www) imgt.cines.fr). IMGT is anintegrated information system that specializes in antibodies, T cellreceptors, and MHC molecules of all vertebrate species. It provides acommon portal to standardized data that include nucleotide and proteinsequences, oligonucleotide primers, gene maps, genetic polymorphisms,specificities, and two-dimensional (2D) and three-dimensional (3D)structures. IMGT includes three sequence databases (IMGT/LIGM-DB,IMGT/MHC-DB, IMGT/PRIMERDB), one genome database (IMGT/GENE-DB), one 3Dstructure database (IMGT/3Dstructure-DB), and a range of web resources(“IMGT Marie-Paule page”) and interactive tools.

V-BASE (I. M. Tomlinson, 2002; World Wide Web (www)mrc-cpe.cam.ac.uk/vbase). V-BASE is a comprehensive directory of allhuman antibody germline variable region sequences compiled from morethan one thousand published sequences. It includes a version of thealignment software DNAPLOT (developed by Hans-Helmar Althaus and WernerMüller) that allows the assignment of rearranged antibody V genes totheir closest germline gene segments.

Antibodies—Structure and Sequence (A. C. R. Martin, 2002; World Wide Web(www) bioinf.org.uk/abs). This page summarizes useful information onantibody structure and sequence. It provides a query interface to theKabat antibody sequence data, general information on antibodies, crystalstructures, and links to other antibody-related information. It alsodistributes an automated summary of all antibody structures deposited inthe Protein Databank (PDB). Of particular interest is a thoroughdescription and comparison of the various numbering schemes for antibodyvariable regions.

AAAAA (A Ho's Amazing Atlas of Antibody Anatomy; A. Honegger, 2001;World Wide Web (www) unizh.ch/-antibody). This resource includes toolsfor structural analysis, modeling, and engineering. It adopts a unifyingscheme for comprehensive structural alignment of antibody andT-cell-receptor sequences, and includes Excel macros for antibodyanalysis and graphical representation.

WAM (Web Antibody Modeling; N. Whitelegg and A. R. Rees, 2001; WorldWide Web (www) antibody.bath.ac.uk). Hosted by the Centre for ProteinAnalysis and Design at the University of Bath, United Kingdom. Based onthe AbM package (formerly marketed by Oxford Molecular) to construct 3Dmodels of antibody Fv sequences using a combination of establishedtheoretical methods, this site also includes the latest antibodystructural information.

Mike's Immunoglobulin Structure/Function Page (M. R. Clark, 2001; WorldWide Web (www) path.cam.ac.uk/˜mrc7/mikeimages.html) These pages provideeducational materials on immunoglobulin structure and function, and areillustrated by many colour images, models, and animations. Additionalinformation is available on antibody humanization and Mike Clark'sTherapeutic Antibody Human Homology Project, which aims to correlateclinical efficacy and anti-immunoglobulin responses with variable regionsequences of therapeutic antibodies.

The Antibody Resource Page (The Antibody Resource Page, 2000; World WideWeb (www) antibodyresource.com). This site describes itself as the“complete guide to antibody research and suppliers.” Links to amino acidsequencing tools, nucleotide antibody sequencing tools, andhybridoma/cell-culture databases are provided.

Humanization bY Design (J. Saldanha, 2000; World Wide Web (www)people.cryst.bbk.ac.uk/-ubcg07s). This resource provides an overview onantibody humanization technology. The most useful feature is asearchable database (by sequence and text) of more than 40 publishedhumanized antibodies including information on design issues, frameworkchoice, framework back-mutations, and binding affinity of the humanizedconstructs.

See also Antibody Engineering Methods and Protocols, Ed. Benny K C Lo,Methods in Molecular Biology™, Human Press. Also at World Wide Web (www)blogsua.com/pdf/antibody-engineering-methods-and-protocolsantibody-engineering-methods-and-protocols.pdf

Any part of this disclosure may be read in combination with any otherpart of the disclosure, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the invention, and are not intended to limit the scope ofwhat the inventors regard as their invention.

EXAMPLES Example 1

High Human Lambda Variable Region Expression in Transgenic MiceComprising Human Lambda Gene Segments Inserted into Endogenous KappaLocus

Insertion of human lambda gene segments from a 1^(st) IGL BAC to the IGKlocus of mouse AB2.1 ES cells (Baylor College of Medicine) was performedto create a chimaeric light chain allele denoted the P1 allele (FIG. 1).The inserted human sequence corresponds to the sequence of humanchromosome 22 from position 23217291 to position 23327884 and comprisesfunctional lambda gene segments Vλ3-1, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3,Jλ6-Cλ6 and Jλ7-Cλ7 (the alleles of Table 13). The insertion was madebetween positions 70674755 and 706747756 on mouse chromosome 6, which isupstream of the mouse Cκ region and 3′Eκ (ie, within 100 kb of theendogenous light chain enhancer) as shown in FIG. 1. The mouse Vκ and Jκgene segments were retained in the chimaeric locus, immediately upstreamof the inserted human lambda DNA. The mouse lambda loci were leftintact. Mice homozygous for the chimaeric P1 locus were generated fromthe ES cells using standard procedures.

A second type of mice were produced (P2 mice) in which more humanfunctional Vλ gene segments were inserted upstream (5′) of human Vλ3-1by the sequential insertion of the BAC1 human DNA and then BAC2 DNA tocreate the P2 allele (the alleles of table 14). The inserted humansequence from BAC2 corresponds to the sequence of human chromosome 22from position 23064876 to position 23217287 and comprises functionallambda gene segments Vλ2-18, Vλ3-16, V2-14, Vλ3-12, Vλ2-11, Vλ3-10,Vλ3-9, Vλ2-8 and Vλ4-3. Mice homozygous for the chimaeric P2 locus weregenerated from the ES cells using standard procedures.

FACS analysis of splenic B cells from the P1 and P2 homozygotes wasperformed to assess lambda versus kappa expression and human lambdaversus mouse lambda expression in the transgenic mice.

Standard 5′-RACE was carried out to analyse RNA transcripts from thelight chain loci in P2 homozygotes.

Light Chain Expression & FACS Analysis

To obtain a single cell suspension from spleen, the spleen was gentlypassage through a 30 μm cell strainer. Single cells were resuspended inPhosphate-Buffered Saline (PBS) supplemented with 3% heat inactivatedfoetal calf serum (FCS).

The following antibodies were used for staining:

Rat anti-mouse lambda (mCλ) phycoerythrin (PE) antibody (SouthernBiotech), rat anti-mouse kappa (mCκ) (BD Pharmingen, clone 187.1)fluorescein isothiocyanate (FITC), anti-human lambda (hCλ) (eBioscience,clone 1-155-2) phycoerythrin (PE), anti-B220/CD45R (eBioscience, cloneRA3-6B2) allophycocyanin (APC). NB: light chains bearing human Cλ wasexpected to have variable regions derived from the rearrangement ofinserted human Vλ and human Jλ. Light chains bearing mouse Cλ wasexpected to have variable regions derived from the rearrangement ofmouse Vλ and Jλ from the endogenous lambda loci.

5×10⁶ cells were added to individual tubes, spun down to remove excessof fluid, and resuspended in fresh 100 μl of PBS+3% FCS. To eachindividual tube the following antibodies were added:

For staining of mλ versus mκ 1 μl of each antibody was added in additionto 1 μl of B220/CD45R antibody. For detection of B cells expressinghuman lambda light chain, the mλ antibody was substituted with hλantibody. Cells were incubated in the dark at 6° C. for 15 minutesfollowed by several washes with fresh PBS+3% FCS to remove unboundantibody. Cells were analysed using fluorescence-activated cell sorting(FACS) analyser from Miltenyi Biotech.

Alive spleenocytes were gated using side scatter (SSC) and forwardscatter (FSC). Within the SSC and FSC gated population, a subpopulationof B220/CD45R (mouse B-cells) was detected using the APC fluorochrome.Single positive B220/CD45R population was further subdivided into a cellbearing either mλ or hλ PE fluorochrome in conjunction with mκ FITCfluorochrome. The percentage of each population was calculated using agating system.

Surprisingly, FACS analysis of splenic B cells from the P1 homozygotesshowed no detectable mouse Cκ expression (FIG. 2), indicating thatinsertion of the human lambda locus DNA from BAC1 interrupts expressionof the endogenous IGK chain.

The strong expression of endogenous Cλ and weak expression of human Cλin the splenic B cells grouped by FACS analysis (mouse Cλ:human Cλ=65:32) in these mice suggest that inserted human IGL sequence, althoughinterrupts the IGK activity, cannot totally compete with the endogenousIGL genes.

The FACS analysis again surprisingly showed no detectable mouse Cκexpression in the P2 homozygotes (FIGS. 3A & B). However, the human Cλgreatly predominates in expressed B cells grouped as mouse or human Cλfollowing FACS analysis (mouse Cλ:human Cλ=15:80 corresponding to aratio of 15 mouse lambda variable regions: 80 human lambda variableregions, ie, 84% human lambda variable regions with reference to thegrouped B-cells—which corresponds to 80% of total B-cells) from the P2homozygotes. While not wishing to be bound by any theory, we suggestthat the inserted human lambda locus sequence from the 2^(nd) BACprovides some advantages to compete with endogenous lambda gene segmentrearrangement or expression.

We analysed human Vλ and Jλ usage in the P2 homozygotes. See FIG. 4which shows the human Vλ usage in P2 homozygotes. The observed usage wassimilar to that seen in humans (as per J Mol Biol. 1997 Apr. 25;268(1):69-77; “The creation of diversity in the human immunoglobulinV(lambda) repertoire”; Ignatovich 0 et an. Further, the human Jλ usagewas similar to that seen in humans (FIG. 5). The Vλ versus Vκ usageanalysis of human Cλ transcripts by sequencing of non-bias 5′-RACE(rapid amplification of cDNA ends) PCR clones showed that among 278clone sequences, only one used Vκ for rearrangement to Jλ (human Jλ),and all others (277 clones) used human Vλ (FIGS. 6 & 7; Vλ2-5 wasdetected at the RNA transcript level, but this is a pseudogene which isusually not picked up by usage a the protein level). While not wishingto be bound by any theory, we suggest that the retained mouse Vκ genesegments essentially cannot efficiently rearrange with the insertedhuman Jλ gene segments because they have the same type of RSSs(recombination signal sequences; see explanation below) and areincompatible for rearrangement (FIG. 8). This result also indicates thatthe inactivation of the endogenous IGK activity and predominateexpression of the inserted human lambda sequence can be achieved withoutfurther modification of the IGK locus, for example, deletion orinversion of endogenous kappa loci gene segments is not necessary, whichgreatly simplifies the generation of useful transgenic mice expressinglight chains bearing human lambda variable regions (ie, variable regionsproduced by recombination of human Vλ and Jλ gene segments).

The arrangement of recombination signal sequences (RSSs) that mediateV(D)J recombination in vivo is discussed, eg, in Cell. 2002 April; 109Suppl:S45-55; “The mechanism and regulation of chromosomal V(D)Jrecombination”; Bassing C H, Swat W, Alt FW (the disclosure of which isincorporated herein by reference). Two types of RSS element have beenidentified: a one-turn RSS (12-RSS) and a two-turn RSS (23-RSS). Innatural VJ recombination in the lambda light chain locus, recombinationis effected between a two-turn RSS that lies 3′ of a V lambda and aone-turn RSS that lies 5′ of a J lambda, the RSSs being in oppositeorientation. In natural VJ recombination in the kappa light chain locus,recombination if effected between a one-turn RSS that lies 3′ of a Vkappa and a two-turn RSS that lies 5′ of a J kappa, the RSSs being inopposite orientation. Thus, generally a two-turn RSS is compatible witha one-turn RSS in the opposite orientation.

Thus, the inventors have demonstrated how to (i) inactivate endogenouskappa chain expression by insertion of human lambda gene segments intothe kappa locus; and (ii) how to achieve very high human lambda variableregion expression (thus providing useful light chain repertoires forselection against target antigen)—even in the presence of endogenouslambda and kappa V gene segments. Thus, the inventors have shown how tosignificantly remove (lambda) or totally remove (kappa) V gene segmentcompetition and thus endogenous light chain expression by the insertionof at least the functional human lambda gene segments comprised by BACs1 and 2. In this example a very high level of human lambda variableregion expression was surprisingly achieved (84% of total lambda chainsand total light chains as explained above).

Example 2

High Human Lambda Variable Region Expression in Transgenic MiceComprising Human Lambda Gene Segments Inserted into Endogenous LambdaLocus

Insertion of human lambda gene segments from the 1st and 2nd IGL BACs tothe lambda locus of mouse AB2.1 ES cells (Baylor College of Medicine)was performed to create a lambda light chain allele denoted the L2allele (FIG. 1). The inserted human sequence corresponds to the sequenceof human chromosome 22 from position 23064876 to position 23327884 andcomprises functional lambda gene segments Vλ2-18, Vλ3-16, V2-14, Vλ3-12,Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3, Vλ3-1, Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3,Jλ6-Cλ6 and Jλ7-Cλ7. The insertion was made between positions 19047551and 19047556 on mouse chromosome 16, which is upstream of the mouse Cλregion and between Eλ4-10 and Eλ3-1 (ie, within 100 kb of the endogenouslight chain enhancers) as shown in FIG. 1. The mouse Vλ and Jλ genesegments were retained in the locus, immediately upstream of theinserted human lambda DNA. The mouse kappa loci were inactivated toprevent kappa chain expression. Mice homozygous for the L2 locus weregenerated from the ES cells using standard procedures.

Using a similar method to that of Example 1, FACS analysis of splenic Bcells from the L2 homozygotes was performed to assess lambda versuskappa expression and human lambda versus mouse lambda expression in thetransgenic mice.

Light Chain Expression & FACS Analysis

The FACS analysis of splenic B-cells in L2 homozygotes under the IGKknockout background (in which Vκ and Jκ gene segments have beenretained) surprisingly showed that expression of human Cλ greatlypredominates in B-cells grouped as mouse or human Cλ following FACSanalysis (mouse Cλ:human Cλ=5:93 corresponding to a ratio of 5 mouselambda variable regions: 93 human lambda variable regions, ie, 95% humanlambda variable regions with reference to the grouped B-cells—whichcorresponds to 93% of total B-cells) (FIG. 9A), demonstrating thatinserted human IGA gene segments within the endogenous IGA locus canoutcompete the endogenous IGA gene segment rearrangement or expression.

Thus, the inventors have demonstrated how to achieve very high humanlambda variable region expression (thus providing useful light chainrepertoires for selection against target antigen)—even in the presenceof endogenous lambda and kappa V gene segments. Thus, the inventors haveshown how to significantly remove endogenous lambda V gene segmentcompetition and thus endogenous lambda light chain expression by theinsertion of at least the functional human lambda gene segmentscomprised by BACs 1 and 2. In this example a very high level of humanlambda variable region expression was surprisingly achieved (95% oftotal lambda chains and total light chains as explained above).

These data indicate that mice carrying either P (Example 1) or L(Example 2) alleles produced by targeted insertion of the functionalgene segments provided by BAC1 and BAC2 can function in rearrangementand expression in mature B cells. These two types of alleles are veryuseful for providing transgenic mice that produce human Ig lambda chainsfor therapeutic antibody discovery and as research tools.

Transgenic Mice of the Invention Expressing Human Lambda VariableRegions Develop Normal Splenic Compartments

In spleen, B cells are characterized as immature (T1 and T2) and mature(M) based on the levels of cell surface markers, IgM and IgD. T1 cellshave high IgM and low IgD. T2 cells have medium levels of both them. Mcells have low IgM but high IgD (FIG. 10). See also J Exp Med. 1999 Jul.5; 190(1):75-89; “B cell development in the spleen takes place indiscrete steps and is determined by the quality of B cellreceptor-derived signals”; Loder F et al.

Using methods similar to those described in Example 3 below, splenicB-cells from the animals were scored for IgD and IgM expression usingFACS. We compared control mice KA/KA (in which endogenous kappa chainexpression has been inactivated, but not endogenous lambda chainexpression) with L2/L2; KA/KA mice (L2 homozyotes). The L2 homozygotessurprisingly showed comparable splenic B-cell compartments to thecontrol mice (FIG. 9B).

Example 3 Assessment of B-Cell and Ig Development in Transgenic Mice ofthe Invention

We observed normal Ig subtype expression & B-cell development intransgenic mice of the invention expressing antibodies with human heavychain variable regions substantially in the absence of endogenous heavyand kappa chain expression.

Using ES cells and the RMCE genomic manipulation methods describedabove, mice were constructed with combinations of the following Ig locusalleles:—

S1F/HA, +/KA=(i) S1F—first endogenous heavy chain allele has one humanheavy chain locus DNA insertion, endogenous mouse VDJ region has beeninactivated by inversion and movement upstream on the chromosome (seethe description above, where this allele is referred to as S1^(inv1));(ii) HA—second endogenous heavy chain allele has been inactivated (byinsertion of an endogenous interrupting sequence); (iii)+—firstendogenous kappa allele is a wild-type kappa allele and (iv) KA—thesecond endogenous kappa allele has been inactivated (by insertion of anendogenous interrupting sequence). This arrangement encodes exclusivelyfor heavy chains from the first endogenous heavy chain allele.

S1F/HA, K2/KA=(i) K2—the first endogenous kappa allele has two kappachain locus DNA insertions between the most 3′ endogenous Jκ and themouse Cκ, providing an insertion of 14 human Vκ and Jκ1-Jκ5; and (ii)KA—the second endogenous kappa allele has been inactivated (by insertionof an endogenous interrupting sequence). This arrangement encodesexclusively for heavy chains comprising human variable regions andsubstantially kappa light chains from the first endogenous kappa allele.

+/HA, K2/KA—this arrangement encodes for mouse heavy chains and humankappa chains.

+/HA, +/KA—this arrangement encodes for mouse heavy and kappa chains—themice only produce mouse heavy and light chains.

In bone marrow, B progenitor populations are characterized based theirsurface markers, B220 and CD43. PreProB cells carry germline IGH andIGK/L configuration and have low B220 and high CD43 on their cellsurface. ProB cells start to initiate VDJ recombination in the IGH locusand carry medium levels of both B220 and CD43. PreB cells carryrearranged IGH VDJ locus and start to initiate light chain VJrearrangement, and have high B220 but low CD43. In spleen, B cells arecharacterized as immature (T1 and T2) and mature (M) based on the levelsof cell surface markers, IgM and IgD. T1 cells have high IgM and lowIgD. T2 cells have medium levels of both them. M cells have low IgM buthigh IgD (FIG. 10). See also J Exp Med. 1991 May 1; 173(5):1213-25;“Resolution and characterization of pro-B and pre-pro-B cell stages innormal mouse bone marrow”; Hardy R R et al and J Exp Med. 1999 Jul. 5;190(1):75-89; “B cell development in the spleen takes place in discretesteps and is determined by the quality of B cell receptor-derivedsignals”; Loder F et al.

Transgenic Mice of the Invention Develop Normal Splenic and BMCompartments

(a) Analysis of the Splenic Compartment

For each mouse, to obtain a single cell suspension from spleen, thespleen was gently passaged through a 30 μm cell strainer. Single cellswere resuspended in Phosphate-Buffered Saline (PBS) supplemented with 3%heat inactivated foetal calf serum (FCS). 5×10⁶ cells were added toindividual tubes, spun down to remove excess of fluid and resuspended infresh 100 μl of PBS+3% FCS. To each individual tube the followingantibodies were added: anti-B220/CD45R (eBioscience, clone RA3-6B2)allophycocyanin (APC), antibody against IgD receptor conjugated withphycoerythrin (PE) (eBioscience, clone 11-26) and antibody against IgMreceptor conjugated with fluorescein isothiocyanate (FITC) (eBioscience,clone 11/41).

For staining of IgM vs IgD, 5×10⁶ cells were used for each staining. Toeach vial containing splenocytes a cocktail of antibodies was addedconsisting of: anti-IgD (PE), anti-IgM (FITC) and anti-B220/CD45R (APC).Cells were incubated at 6° C. for 15 minutes, washed to remove excessunbound antibodies and analysed using a fluorescence-activated cellsorting (FACS) analyser from Miltenyi Biotech. B-cells were gated asB220^(HIGH) IgM^(HIGH) IgD^(LOW) (ie, B220⁺ IgM⁺ IgD⁻) for T1population, B220^(HIGH) IgM^(HIGH) IgD^(HIGH) (B220⁺ IgM⁺ IgD⁺) for T2population and B220^(HIGH) IgM^(LOW) IgD^(HIGH) (B220⁺ IgM⁻ Ig1D⁺) for Mpopulation. Percentage of cells was calculated using gating system. Weused gates to identify and define subsets of cell populations on plotswith logarithmic scale. Before gates are applied a single stain antibodyfor each fluorochrome is used to discriminate between a positive (highintensity fluorochrome) and negative (no detectable intensityfluorchrome) population. Gates are applied based on fluorochromeintensities in the same manner to all samples. The single stains were:

IgD-PE

IgM-FITC

B220-APC

Alive spleenocytes were gated using side scatter (SSC) and forwardscatter (FSC). Within the SSC and FSC gated population, a subpopulationof B220/CD45R positive cells (mouse B-cells) was detected using the APCfluorochrome. The single positive B220/CD45R population was furthersubdivided into a cell bearing either IgM fluorescein isothiocyanate(FITC) or IgD fluorochrome in conjunction with Mκ FITC fluorochrome. Thepercentage of each population was calculated using gating system. Thesplenic B-Cell compartments in the mice of the invention are normal (ie,equivalent to the compartments of mice expressing only mouse antibodychains).

(b) Bone Marrow B Progenitor Analysis

To obtain a single cell suspension from bone marrow for each mouse, thefemur and tibia were flushed with Phosphate-Buffered Saline (PBS)supplemented with 3% heat inactivated foetal calf serum (FCS). Cellswere further passage through a 30 μm cell strainer to remove bone piecesor cell clumps. Cells were resuspended in cold PBS supplemented with 3%serum. 2×10⁶ cells were added to individual tubes, spun down to removeexcess of buffer, and resuspended in fresh 100 μl of PBS+3% FCS. To eachindividual tube the following antibodies were added: anti-Leukosialin(CD43) fluorescein isothiocyanate (FITC) (eBioscience, clone eBioR2/60)and anti-B220/CD45R (eBioscience, clone RA3-6B2) allophycocyanin (APC).Cells were incubated in the dark at 6° C. for 15 minutes followed byseveral washes with fresh PBS+3% FCS to remove unbound antibody. Cellswere analysed using a fluorescence-activated cell sorting (FACS)analyser from Miltenyi Biotech. Alive bone marrow cells were gated usingside scatter (SSC) and forward scatter (FSC). We used gates to identifyand define subsets of cell populations on plots with logarithmic scale.Before gates are applied a single stain antibody for each fluorochromeis used to discriminate between a positive (high intensity fluorochrome)and negative (no detectable intensity fluorchrome) population. Gates areapplied based on fluorochrome intensities in the same manner to allsamples. The single stains were:

B220-APC

CD43-FITC

Within the alive population a double population of B220/CD45R and CD43positive cells was identified as a pre-B, pro-B and pre-pro B cells. Thesplenic B-Cell compartments in the mice of the invention are normal (ie,equivalent to the compartments of mice expressing only mouse antibodychains).

Transgenic Mice of the Invention Develop Normal Ig Expression

Quantification of Serum IgM and IgG

96-well NUNC plates were coated initially with a capture antibody (goatanti-mouse Fab antibody at 1 μg/ml) overnight at 4° C.). The IgG platesused anti-Fab, (M4155 Sigma) and the IgM plates used anti-Fab (OBT1527AbD Serotec). Following three washes with phosphate buffer saline (PBS)containing 0.1% v/v Tween20, plates were blocked with 200 μl of PBScontaining 1% w/v bovine serum albumin (BSA) for 1 hour at roomtemperature (RT). The plates were washed three times as above and then50 μl of standards (control mouse isotype antibodies, IgG1 (M9269Sigma), IgG2a (M9144 Sigma), IgG2b (M8894 sigma), IgM (M3795 Sigma) orserum samples diluted in PBS with 0.1% BSA were added to each well, andincubated for 1 hour at RT. After washing three times as above 100 μl ofdetection antibody (goat anti-mouse isotype specificantibody-horseradish peroxidase conjugated, 1/10000 in PBS with 0.1%Tween) (anti-mouse IgG1 (STAR132P AbD Serotec), anti-mouse IgG2a(STAR133P AdD Serotec), anti-mouse IgG2b (STAR134P AbD Serotec) andanti-mouse IgM (ab97230 Abcam) were added into each well and incubatedfor 1 hour at RT. The plates were washed three times as above anddeveloped using tetramethylbenzidine substrate (TMB, Sigma) for 4-5minutes in the dark at RT. Development was stopped by adding 50 μl/wellof 1 M sulfuric acid. The plates were read with a Biotek Synergy HTplate reader at 450 nm.

Conclusion

Inversion of endogenous V_(H)-D-J_(H) following the human IGH BACinsertion results in inactivation of rearrangement of endogenous V_(H)to inserted human D-J_(H). The inventors observed, however, thatsurprisingly the inactivation of endogenous heavy chain expression doesnot change the ratio of B-cells in the splenic compartment (FIG. 11) orbone marrow B progenitor compartment (FIG. 12) and the immunoglobulinlevels in serum are normal and the correct Ig subtypes are expressed(FIG. 13). This was shown in mice expressing human heavy chain variableregions with mouse light chains (FIGS. 11A and 12A) as well as in miceexpressing both human heavy chain variable regions and human light chainvariable regions (FIGS. 11B and 12B). These data demonstrate thatinserted human IGH gene segments (an insertion of at least human V_(H)gene segments V_(H)2-5, 7-4-1, 4-4, 1-3, 1-2, 6-1, and all the human Dand J_(H) gene segments D1-1, 2-2, 3-3, 4-4, 5-5, 6-6, 1-7, 2-8, 3-9,5-12, 6-13, 2-15, 3-16, 4-17, 6-19, 1-20, 2-21, 3-22, 6-25, 1-26 and7-27; and J1, J2, J3, J4, J5 and J6) are fully functional in the aspectof rearrangement, BCR signalling and B cell maturation. Functionality isretained also when human light chain VJ gene segments are inserted toprovide transgenic light chains, as per the insertion used to create theK2 allele. This insertion is an insertion comprising human gene segmentsVκ2-24, Vκ3-20, Vκ1-17, Vκ1-16, Vκ3-15, Vκ1-13, Vκ1-12, Vκ3-11, Vκ1-9,Vκ1-8, Vκ1-6, Vκ1-5, Vκ5-2, Vκ4-1, Jκ1, Jκ2, Jκ3, Jκ4 and Jκ5. Greaterthan 90% of the antibodies expressed by the S1F/HA; K2/KA mice comprisedhuman heavy chain variable regions and human kappa light chain variableregions. These mice are, therefore, very useful for the selection ofantibodies having human variable regions that specifically bind humanantigen following immunisation of the mice with such antigen. Followingisolation of such an antibody, the skilled person can replace the mouseconstant regions with human constant regions using conventionaltechniques to arrive at totally human antibodies which are useful asdrug candidates for administration to humans (optionally followingmutation or adaptation to produce a further derivative, eg, with Fcenhancement or inactivation or following conjugation to a toxic payloador reporter or label or other active moiety).

A further experiment was carried out to assess the IgG and IgM levelsand relative proportions in transgenic mice of the invention thatexpress antibodies that have human heavy and light (kappa) variableregions (S1F/HA, K2/KA mice; n=15). These were compared against 12 miceexpressing only mouse antibody chains (+/HA, +/KA (n=6) and wild-typemice (WT; n=6)). The results are tabulated below (Table 19) and shown inFIG. 14.

It can be seen that the mice of the invention, in which essentially allheavy chain variable regions are human heavy chain variable regions,expressed normal proportions of IgM and IgG subtypes, and also total IgGrelative to IgM was normal.

TABLE 19 IgG1 IgG2a IgG2b IgM Total IgG + IgM (μg/mL) (μg/mL) (μg/mL)(μg/mL) (μg/mL) KMCB22.1a 30.5 38.3 49.9 1.7 224.4 1.8 343.1 KMCB 19.1d103.6 181.2 85.6 1.9 351.7 1.10 722.1 S1F/HA, K2/KA KMCB 19.1h 191.4456.6 383.3 1.11 643.2 1.12 1674.6 S1F/HA, K2/KA KMCB 20.1a 53.6 384.4249.7 1.13 427.1 1.14 1114.7 S1F/HA, K2/KA KMCB 20.1c 87.3 167.0 125.71.15 422.1 1.16 802.1 S1F/HA, K2/KA KMCB 20.1f 55.4 177.2 95.6 1.17295.7 1.18 623.9 S1F/HA, K2/KA KMCB22.1f 61.1 56.3 111.4 1.19 245.8 1.20474.5 S1F/HA, K2/KA KMCB23.1c 71.4 70.7 80.5 1.21 585.4 1.22 808.0S1F/HA, K2/KA KMCB23.1d 65.4 148.7 187.4 1.23 255.4 1.24 657.0 S1F/HA,K2/KA KMCB24.1f 60.0 56.6 150.5 1.25 294.8 1.26 561.9 S1F/HA, K2/KAKMCB13.1a 101.2 200.5 269.8 1.27 144.1 1.28 715.7 S1F/HA, K2/KAKMCB13.1d 124.5 117.5 246.6 1.29 183.2 1.30 671.9 S1F/HA, K2/KAKMCB17.1f 58.3 174.2 1.31 116.2 1.32 218.1 1.33 566.8 S1F/HA, K2/KAKMCB14.1a 51.9 46.5 27.9 1.34 222.2 1.35 348.6 S1F/HA, K2/KA KMCB14.1b11.5 54.2 48.5 1.36 194.4 1.37 308.6 S1F/HA, K2/KA KMCB19.1e +/HA, 233.06.7 465.6 1.38 420.9 1.39 1126.3 +/KA KMCB19.1f +/HA, 154.3 4.6 610.21.40 435.7 1.41 1204.8 +/KA KMCB19.1l +/HA, 113.5 1.1 246.8 1.42 374.61.43 736.0 +/KA KMCB20.1e +/HA, 561.0 4.3 614.3 1.44 482.1 1.45 1661.7+/KA KMCB13.1e +/HA, 439.3 17.1 584.1 1.46 196.9 1.47 1237.3 +/KAKMCB14.1c +/HA, 93.4 1.3 112.0 1.48 106.8 1.49 313.6 +/KA KMWT 212.9155.2 104.6 1.50 233.7 1.51 706.4 1.3c WT KMWT 297.1 203.2 144.6 1.52248.6 1.53 893.5 1.3d WT KMWT 143.1 174.2 619.1 1.54 251.8 1.55 1188.21.3e WT KMWT 218.8 86.8 256.1 1.56 294.8 1.57 856.4 1.3f WT KMWT 150.2114.2 114.7 1.58 225.6 1.59 604.7 1.3b WT KMWT 125.9 335.5 174.6 1.60248.9 1.61 884.9 3.1e WT

Example 4 Assessment of Kappa:Lambda Ratio & Splenic B-Cell Compartmentsin Transgenic Mice of the Invention

Mice comprising the following genomes were obtained.

WT/WT=wild-type;

KA/KA=each endogenous kappa allele has been inactivated; and theendogenous lambda loci are left intact;

K3F/K3F=each endogenous kappa allele has three kappa chain locus DNAinsertions between the 3′ most endogenous Jκ and the mouse Cκ, providinginsertion of human V gene segments Vκ2-40, Vκ1-39, Vκ1-33, Vκ2-30,Vκ2-29, Vκ2-28, Vκ1-27, Vκ2-24, Vκ3-20, Vκ1-17, Vκ1-16, Vκ3-15, Vκ1-13,Vκ1-12, Vκ3-11, Vκ1-9, Vκ1-8, Vκ1-6, Vκ1-5, Vκ5-2 and Vκ4-1 and human Jgene segments Jκ1, Jκ2, Jκ3, Jκ4 and Jκ5 (the human V gene segmentsbeing 5′ of the human J gene segments); each endogenous kappa VJ hasbeen inactivated by inversion and movement upstream on the chromosome;and the endogenous lambda loci are left intact;

L2/L2=as described in Example 2 (L2 homozygotes where human lambdavariable region DNA has been inserted into the endogenous lambda loci;the endogenous kappa loci are left intact);

L2/L2; KA/KA=as L2/L2 but the endogenous kappa alleles have beeninactivated (by insertion of an endogenous interrupting sequence=KA);

L3/L3; KA/KA=as L2/L2; KA/KA but supplemented by a third human lambdavariable region DNA insertion 5′ of the second lambda DNA insertion inthe endogenous lambda loci such that the following human lambda genesegments are inserted between 3′ most endogenous Jλ and the mouse Cλ:human V gene segments Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19,Vλ2-18, Vλ3-16, Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, V3-9, Vλ2-8, Vλ4-3 andVλ3-1, human J and C gene segments Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6and Jλ7-Cλ7 (non-functional segments Jλ4-Cλ4, Jλ5-Cλ5 were alsoincluded), thus providing an insertion corresponding to coordinates22886217 to 23327884 of human chromosome 22 inserted immediately afterposition 19047551 on mouse chromosome 16;

S3F/HA; KA/KA; L3/L3=first endogenous heavy chain allele has three humanheavy chain variable region DNA insertions between the 3′ mostendogenous J_(H) and the E_(μ), providing insertion of human genesegments V_(H)2-26, V_(H)1-24, V_(H)3-23, V_(H)3-21, V_(H)3-20,V_(H)1-18, V_(H)3-15, V_(H)3-13, V_(H)3-11, V_(H)3-9, V_(H)1-8,V_(H)3-7, V_(H)2-5, V_(H)7-4-1, V_(H)4-4, V_(H)1-3, V_(H)1-2, V_(H)6-1,D1-1, D2-2, D3-9, D3-10, D4-11, D5-12, D6-13, D1-14, D2-15, D3-16,D4-17, D5-18, D6-19, D1-20, D2-21, D3-22, D4-23, D5-24, D6-25, D1-26,D7-27, J_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)5 and J_(H)6 (in the order:human V gene segments, human D gene segments and human J gene segments);the endogenous heavy chain VDJ sequence has been inactivated byinversion and movement upstream on the chromosome; and the endogenouslambda loci are left intact; the second endogenous heavy chain allelehas been inactivated by insertion of an endogenous interruptingsequence=HA); the endogenous kappa alleles have been inactivated(=KA/KA); and the endogenous lambda alleles have been modified byinsertion of human lambda variable region DNA (=L3/L3);

P2/WT=P2 allele (human lambda variable region DNA as described inExample 1) at one endogenous kappa locus; the other endogenous kappalocus left intact; both endogenous lambda loci left intact;

P2/P2=see Example 14; both endogenous lambda loci left intact;

P2/K2=P2 allele at one endogenous kappa locus; the other endogenouskappa locus having two DNA insertions between the 3′ most endogenous Jκand the mouse Cκ, providing insertion of human V gene segments Vκ2-24,Vκ3-20, Vκ1-17, Vκ1-16, Vκ3-15, Vκ1-13, Vκ1-12, Vκ3-11, Vκ1-9, Vκ1-8,Vκ1-6, Vκ1-5, Vκ5-2 and Vκ4-1 and human J gene segments Jκ1, Jκ2, Jκ3,Jκ4 and Jκ5 (the human V gene segments being 5′ of the human J genesegments); both endogenous lambda loci left intact;

P3/K3F=as one endogenous kappa locus having an insertion between thefollowing human lambda gene segments are inserted between the 3′ mostendogenous Jκ and the mouse Cκ, providing insertion of human V genesegments Vλ3-27, Vλ3-25, Vλ2-23, Vλ3-22, Vλ3-21, Vλ3-19, Vλ2-18, Vλ3-16,Vλ2-14, Vλ3-12, Vλ2-11, Vλ3-10, Vλ3-9, Vλ2-8, Vλ4-3 and Vλ3-1, human Jand C gene segments Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3, Jλ6-Cλ6 and Jλ7-Cλ7 (nonfunctional segments Jλ4-Cλ4, JIBS-CICS were also included), thusproviding an insertion corresponding to coordinates 22886217 to 23327884of human chromosome 22 inserted immediately after position 70674755 onmouse chromosome 6; the other endogenous kappa locus having the K3Fallele described above (human V and J kappa gene segments inserted);both endogenous lambda loci left intact;

P2/P2; L2/WT=As P2/P2 but wherein one endogenous lambda locus has the L2allele (human lambda V and J gene segments inserted) and the otherendogenous lambda locus is wild-type; and

P2/P2; L2/L2=homozygous for P2 and L2 alleles at endogenous kappa andlambda loci respectively.

FACS analysis of splenic B-cells (as described above) was carried outand proportions of light chain expression were determined. We alsodetermined the proportions of T1, T2 and mature (M) splenic B-cells andcompared with wild-type mice, in order to assess whether or not weobtained normal splenic B-cell compartments in the transgenic mice. Theresults are shown in Tables 20 and 21. We also assessed the proportionof B220 positive cells as an indication of the proportion of B-cells inthe splenic cell samples.

TABLE 20 Comparisons With Mice With Human Lambda Variable Region InsertsAt Endogenous Lambda Locus IGL percentage ml ml Splenic B-cellcompartment Genotype B220 1.1 Gκ 1.2 Gλ 1.3 hIGλ T1 T2 M WT/WT   20%  90%  3.80% 1.4 1.5    16% 1.6 16.5 1.7 57.50% (n = 2) KA/KA 13.60% 0.28% 68.50% 1.8     0% 1.9    33% 1.10     9% 1.11    41% (n = 2)K3F/K3F   20%   83%     7% 1.12 1.13    16% 1.14 15.50% 1.15    58% (n =2) L2/L2 (n = 2) 17.80% 91.60%  1.60% 1.16  6.50% 1.17 21.50% 1.18   10% 1.19    50% L2/L2;KA/K  9.10%     0%     5% 1.20    93% 1.21   28% 1.22     7% 1.23    44% A (n = 1) L3/L3;KA/K 16.90%  0.10%  4.50%1.24 93.20% 1.25 17.40% 1.26 13.10% 1.27 53.90% A (n = 2) S3F/HA; KA/    19%  0.20%  3.80% K;L3/L3 1.28    98% 1.29 15.50% 1.30    19% 1.3153.20% (n = 1)

TABLE 21 Mice With Human Lambda Variable Region Inserts At EndogenousKappa Locus IGL Percentage Splenic B-cell compartment Genotype B220 1.1mIGκ 1.2 mIGλ 1.3 hIGλ T1 T2 M P2/WI N.D   90%  4.20% 1.4  6.55% 1.517.30% 1.6  8.90% 1.7 52.50% (n = 2) P2/P2 14.80%  0.20%    15% 1.8   76% 1.9 27.50% 1.10    12% 1.11    42% (n = 2) P2/K2 18.20% 78.80% 7.90% 1.12 15.60% 1.13 19.50% 1.14    12% 1.15    50% (n = 2) P3/K3F18.40% 64.80% 11.60% 1.16 19.40% 1.17 11.80% 1.18 18.40% 1.19 56.10% (n= 2) P2/P2; L2/WI 20.40%  0.05%  8.50% 1.20    94% 1.21 13.10% 1.2216.10% 1.23 59.90% (n = 2) P2/P2; L2/L2 12.70% 0.07%  5.10% 1.24 95.40%1.25 13.40% 1.26 13.80% 1.27 57.30% (n = 2)

CONCLUSIONS

As demonstrated by L2/L2; KA/KA and L3/L3; KA/KA, the human lambdavariable region DNA insertions at the endogenous lambda locus (with anendogenous kappa knockout) displayed predominate expression of lightchains bearing human lambda variable regions (indicated by theexpression of Cλ-positive chains at around 93%). This surprisinglyoccurs even though endogenous mouse lambda variable region DNA is stillpresent, indicating that the inserted human lambda variable region DNAcan outcompete endogenous IGA rearrangement.

Furthermore, mice having the human V and J gene segments present in thehomozygous L3 insertion produce B-cells (B220 positive cells) at aproportion that is similar to wild-type and additionally produce anormal proportion or percentage of mature splenic B-cells (ie, similarto wild-type). This is confirmed not only by the L3/L3; KA/KA mice, butalso was observed for S3F/HA; KA/KA; L3/L3, which also comprises achimaeric (human-mouse) IgH locus.

Also, we observed that mice having the human V and J gene segmentspresent in the homozygous K3F insertion produce B-cells (B220 positivecells) at a proportion that is similar to wild-type and additionallyproduce a normal proportion or percentage of mature splenic B-cells (ie,similar to wild-type).

Mice having the human V and J gene segments present in the homozygous P2insertion at the endogenous kappa locus showed high expression of lightchains comprising human lambda variable regions (as indicated by anobserved proportion of 76%). We could skew to an even higher percentageoverall by combining insertion of human lambda V and J gene segments atboth the endogenous kappa and lambda loci (see P2/P2; L2/WT at around94% and P2/P2; L2/L2 at around 95%). Furthermore, mice comprising thehuman V and J gene segment arrangement of P2/P2; L2/L2 produce a normalproportion or percentage of mature splenic B-cells (ie, similar towild-type).

When human lambda V and J gene segments were inserted at one endogenouskappa locus and the other endogenous kappa locus comprised an insertionof human kappa V and J gene segments, we obtained mice that couldexpress light chains comprising lambda variable regions and also lightchains comprising kappa variable regions. Surprisingly observed that wecould raise the proportion of light chains comprising lambda variableregions above that seen in a wild-type mouse where only 5% or less oflight chains typically comprise lambda variable regions. We observed aproportion of around 22% for the P2/K2 genotype and around 31% for theP3/K3F genotype. The proportion observed with the latter genotypeapproximates that seen in a human where typically around 60% of lightchains comprise kappa variable regions and around 40% of light chainscomprise lambda variable regions. Also in the P2/K2 and P3/K3F cases,the mice produced a normal proportion of B-cells as compared withwild-type mice. Furthermore, mice comprising the human V and J genesegment arrangement of P3/K3F produce a normal proportion or percentageof mature splenic B-cells (ie, similar to wild-type).

Example 5

Mouse were generated that comprised the specific IgH alleles listed inTable 3; and the specific IgL alleles listed in Tables 10 or 11. Micewere immunised with target antigens and antigen-specific antibodies wereisolated. Antibodies were assessed for binding specificity, maturation(ie, extent of junctional and somatic mutation versus germline genesegment sequences) and binding kinetics. Corresponding B-cells were alsoobtained and in some cases hybridomas produced that express the selectedantibodies.

Selected antibodies are summarised in Table 22. Binding kinetics of someof these were determined as follows.

Binding Kinetics Determination

An anti-mouse IgG capture surface was created on a GLM Biosensor™ chipby primary amine coupling using GE Healthcare anti-mouse IgG(BR-1008-38). Test antibodies as set out in Table 22 were captured onthis surface and the respective antigen was passed over the captured Abat the concentrations indicated. An injection of buffer (i.e. 0 nM ofantigen) was used to double reference the binding curves, and the datawas fitted to the 1:1 model inherent to the ProteOn XPR36™ analysissoftware. Regeneration of the capture surface was carried out using 10mM glycine, pH1.7. The assay was run at 25° C. and using HBS-EP asrunning buffer.

Target 1: a multi-subunit human protein

Target 2: a bacterial cytotoxin

Target 3: a different multi-subunit human protein

Target 4: a protein expressed as a transmembrane protein on human cells

Target 1 mAb1.1

Single concentration TARGET 1 (256 nM), anti-mouse capture

ka kd KD 3.85E+05 3.22E−05 83 pM

(Apparent affinity since multi-subunit target)

Target 2 mAb2.1

TARGET 2 at 256, 64, 16, 4 and 1 nM; results of 3 experiments:—

Experiment 1

ka kd KD 1.40E+04 1.83E−05 1.300 nM

Experiment 2

ka kd KD 2.76E+04 3.23E−05 1.170

Experiment 3

Couldn't resolve off-rate—indicating extremely tight binding beyonddetectable limits.

Target 3 mAb3.1

TARGET 3 at 256, 64, 16, 4 and 1 nM

ka kd KD 4.00E+05 2.34E−04 0.59 nM

(Apparent affinity since multi-subunit target)

Target 3 mAb3.2

TARGET 3 at 256, 64, 16, 4 and 1 nM

ka kd KD 3.86E+05 2.57E−04 0.67

(Apparent Affinity Since Multi-Subunit Target)

Target 3 mAb3.3

TARGET 3 at 256, 64, 16, 4 and 1 nM

Unable to resolve Off-rate, extremely tight binding

(Apparent Affinity Since Multi-Subunit Target)

In conclusion, the present invention provides for in vivoaffinity-matured antibodies with human variable domains that canexpressed in in vivo systems, and specifically bind target antigens withvery good affinities, on and off-rates. The invention thus provides forantibodies that are useful for human medicine, as well as non-humanvertebrates, cells (eg, B-cells and hybridomas) for producing suchantibodies.

Example 6

The S (heavy), K(kappa into kappa locus), L (lambda into lambda locus)and P (lambda into kappa locus) lines used to generate the data in theexamples used the alleles of Tables 1 to 18 and demonstrated that suchcollections of alleles can produce the surprising results shown (eg,good B cell compartments, high human lambda V region expression,desirable lambda:kappa ratio in a mouse and normal repertoire of IgHisotypes). The isolated antibodies were all based on the alleles listedin Table 1 to 18 above. All had V domains with mouse AID and TdT patternmutation.

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

TABLE 22 AA- knon- kAA- Hybridoma nonGerm- Muta- Germ- Muta- Sequences vd j 1.28 lineAA¹ 1.29 tions² 1.30 kv 1.31 kj 1.32 lineAA¹ 1.33 tions²TARGET 1: mAb1.1 IGHV7- IGHD3- IGHJ6* 1.34 2 1.35 0 1.36 IGKV2- 1.37IGKJ4* 1.38 0 1.39 0 4- 16*02 02 28*01 1 1*01 mAb1.2 IGHV4- IGHD3-IGHJ6* 1.40 3 1.41 0 1.42 IGKV1D- 1.43 IGKJ4* 1.44 0 1.45 0 4*02 10*0102 13*d01 1 TARGET 2: mAb2.1 IGHV1- IGHD3- IGHJ6* 1.46 6 1.47 9 1.48IGKV1- 1.49 IGKJ4* 1.50 1 1.51 0 3*01 10*01 02 12*01 1 TARGET 3: mAb3.1IGHV3- IGHD3- IGHJ6* 1.52 3 1.53 5 1.54 IGKV1D- 1.55 IGKJ4* 1.56 0 1.574 13*01 9*01 02 12*02 1 mAb3.2 IGHV3- IGHD3- IGHJ6* 1.58 3 1.59 5 1.60IGKV1D- 1.61 IGKJ4* 1.62 0 1.63 4 13*01 9*01 02 12*02 1 mAb3.3 IGHV4-IGHD3- IGHJ6* 1.64 2 1.65 7 1.66 IGKV1D- 1.67 IGKJ4* 1.68 3 1.69 3 4*0210*01 02 13d*01 1 Bcell Tech AA- knon- kAA- Sequences nonGerm- Muta-Germ- Muta- id v d j 1.70 lineAA¹ 1.71 tions² 1.72 kv 1.73 kj 1.74lineAA¹ 1.75 tions² TARGET 1: mAb1.3 IGHV3- IGHV3- IGHJ6* 1.76 5 1.77 01.78 IGKV3- 1.79 IGKJ4* 1.80 0 1.81 0 13*01 13*01 02 20*01 1 AA- knon-kAA- nonGerm- Muta- Germ- Muta- id v d j 1.82 lineAA¹ 1.83 tions² 1.84kv 1.85 kj 1.86 lineAA¹ 1.87 tions² TARGET 3: mAb3.4 IGHV3- IGHD3-IGHJ6* 1.88   9 1.89 8 1.90  IGKV1- 1.91  IGKJ4* 1.92  1 1.93  1 23*0422*01 02 17*01 1 mAb3.5 IGHV3- IGHD3- IGHJ6* 1.94  10 1.95 5 1.96 IGKV1- 1.97  IGKJ4* 1.98  1 1.99  0 23*04 22*01 02 17*01 1 mAb3.6 IGHV3-IGHD3- IGHJ6* 1.100  6 1.101 2 1.102 IGKV1D- 1.103 IGKJ4* 1.104 1 1.1053 7*01 9*01 02 39*01 1 mAb3.7 IGHV3- IGHD3- IGHJ6* 1.103  9 1.107 51.108 IGKV1- 1.109 IGKJ4* 1.110 1 1.111 1 23*04 22*01 02 17*01 1 mAb3.8IGHV3- IGHD3- IGHJ6* 1.112  7 1.113 6 1.114 IGKV1D- 1.115 IGKJ4* 1.116 21.117 4 13*01 10*01 02 39*01 1 mAb3.9 IGHV3- IGHD3- IGHJ6* 1.118  31.119 3 1.120 IGKV3- 1.121 IGKJ4* 1.122 0 1.123 5 13*01 10*01 02 11*01 1AA- knon- kAA- nonGerm- Muta- Germ- Muta- id v d j 1.124 lineAA¹ 1.125tions² 1.126 kv 1.127 kj 1.128 lineAA¹ 1.129 tions² TARGET 4: mAb4.1IGHV4- IGHD3- IGHJ6* 1.130 7 1.131 7 1.132 IGKV1D- 1.133 IGKJ4* 1.44 11.135 1 4*02 9*01 02 16*01 1 mAb4.2 IGHV3- IGHD3- IGHJ6* 1.136 6 1.137 41.138 IGKV1- 1.139 IGKJ4* 1.44 1 1.141 1 20*d01 10*01 02 9*d01 1 [Allgene segments are human] ¹nonGermlineAA: number of non-germline aminoacids introduced into VH-D or D-JH junctions or into VL-JL junctions²AAMutations: number of AA mutations in V and J region (CDRH3 or CDRL3region excluded)

Sequences for examples of gene segments in accordance with the inventionare set out below.

IGLC7*01ggtcagcccaaggctgccccctcggtcactctgttcccaccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcgtaagtgacttctacccgggagccgtgacagtggcctggaaggcagatggcagccccgtcaaggtgggagtggagaccaccaaaccctccaaacaaagcaacaacaagtatgcggccagcagctacctgagcctgacgcccgagcagtggaagtcccacagaagctacagctgccgggtcacgcatgaagggagcaccgtggagaagacagtggcccctgcagaatgctctIGLJ7*01 tgctgtgttcggaggaggcacccagctgaccgtcctcg IGLC6*01ggtcagcccaaggctgccccatcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgcctgatcagtgacttctacccgggagctgtgaaagtggcctggaaggcagatggcagccccgtcaacacgggagtggagaccaccacaccctccaaacagagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctgcagaatgttcaIGLC6*04gtcagcccaaggctgccccatcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgcctgatcagtgacttctacccgggagctgtgaaagtggcctggaaggcagatggcagccccgtcaacacgggagtggagaccaccacaccctccaaacagagcaacaacaagtacgcggccagcagctagctacctgagcctgacgcctgagcagtggaagtcccacagaagctacagttgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctgcagaatgctctIGLJ6*01 taatgtgttcggcagtggcaccaaggtgaccgtcctcg IGLC3*03ggtcagcccaaggctgccccctcggtcactctgttcccaccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgagcagtggaagtcccacaaaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttcaIGLJ3*02 ttgggtgttcggcggagggaccaagctgaccgtcctag IGLC2*02ggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttcaIGLJ2*01 tgtggtattcggcggagggaccaagctgaccgtcctag IGLC1*02ggtcagcccaaggccaaccccactgtcactctgttcccgccctcctctgaggagctccaagccaacaaggccacactagtgtgtctgatcagtgacttctacccgggagctgtgacagtggcctggaaggcagatggcagccccgtcaaggcgggagtggagaccaccaaaccctccaaacagagcaacaacaagtacgcggccagcagctacctgagcctgacgcccgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttcaIGLJ1*01 ttatgtcttcggaactgggaccaaggtcaccgtcctag IGLV3-1*01gatccgtggcctcctatgagctgactcagccaccctcagtgtccgtgtccccaggacagacagccagcatcacctgctctggagataaattgggggataaatatgcttgctggtatcagcagaagccaggccagtcccctgtgctggtcatctatcaagatagcaagcggccctcagggatccctgagcgattctctggctccaactctgggaacacagccactctgaccatcagcgggacccaggctatggatgaggctgactattactgtcaggcgtgggacagcagcactgca IGLV4-3*01ctgcctgtgctgactcagcccccgtctgcatctgccttgctgggagcctcgatcaagctcacctgcaccctaagcagtgagcacagcacctacaccatcgaatggtatcaacagagaccagggaggtccccccagtatataatgaaggttaagagtgatggcagccacagcaagggggacgggatccccgatcgcttcatgggctccagttctggggctgaccgctacctcaccttctccaacctccagtctgacgatgaggctgagtatcactgtggagagagccacacgattgatggccaagtcggttgagcIGLV2-8*01cagtctgccctgactcagcctccctccgcgtccgggtctcctggacagtcagtcaccatctcctgcactggaaccagcagtgacgttggtggttataactatgtctcctggtaccaacagcacccaggcaaagcccccaaactcatgatttatgaggtcagtaagcggccctcaggggtccctgatcgcttctctggctccaagtctggcaacacggcctccctgaccgtctctgggctccaggctgaggatgaggctgattattactgcagctcatatgcaggcagcaacaatttc IGLV3-9*01tcctatgagctgactcagccactctcagtgtcagtggccctgggacagacggccaggattacctgtgggggaaacaacattggaagtaaaaatgtgcactggtaccagcagaagccaggccaggcccctgtgctggtcatctatagggatagcaaccggccctctgggatccctgagcgattctctggctccaactcggggaacacggccaccctgaccatcagcagagcccaagccggggatgaggctgactattactgtcaggtgtgggacagcagcactgca IGLV3-10*01tcctatgagctgacacagccaccctcggtgtcagtgtccccaggacaaacggccaggatcacctgctctggagatgcattgccaaaaaaatatgcttattggtaccagcagaagtcaggccaggcccctgtgctggtcatctatgaggacagcaaacgaccctccgggatccctgagagattctctggctccagctcagggacaatggccaccttgactatcagtggggcccaggtggaggatgaagctgactactactgttactcaacagacagcagtggtaatcatag IGLV2-11*01cagtctgccctgactcagcctcgctcagtgtccgggtctcctggacagtcagtcaccatctcctgcactggaaccagcagtgatgttggtggttataactatgtctcctggtaccaacagcacccaggcaaagcccccaaactcatgatttatgatgtcagtaagcggccctcaggggtccctgatcgcttctctggctccaagtctggcaacacggcctccctgaccatctctgggctccaggctgaggatgaggctgattattactgctgctcatatgcaggcagctacactttc IGLV3-12*02tcctatgagctgactcagccacactcagtgtcagtggccacagcacagatggccaggatcacctgtgggggaaacaacattggaagtaaagctgtgcactggtaccagcaaaagccaggccaggaccctgtgctggtcatctatagcgatagcaaccggccctcagggatccctgagcgattctctggctccaacccagggaacaccgccaccctaaccatcagcaggatcgaggctggggatgaggctgactattactgtcaggtgtgggacagtagtagtgatcatcc IGLV2-14*01cagtctgccctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaaccagcagtgacgttggtggttataactatgtctcctggtaccaacagcacccaggcaaagcccccaaactcatgatttatgaggtcagtaatcggccctcaggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgaccatctctgggctccaggctgaggacgaggctgattattactgcagctcatatacaagcagcagcactctc IGLV3-16*01tcctatgagctgacacagccaccctcggtgtcagtgtccctaggacagatggccaggatcacctgctctggagaagcattgccaaaaaaatatgcttattggtaccagcagaagccaggccagttccctgtgctggtgatatataaagacagcgagaggccctcagggatccctgagcgattctctggctccagctcagggacaatagtcacattgaccatcagtggagtccaggcagaagacgaggctgactattactgtctatcagcagacagcagtggtacttatcc IGLV2-18*01cagtctgccctgactcagcctccctccgtgtccgggtctcctggacagtcagtcaccatctcctgcactggaaccagcagtgacgttggtagttataaccgtgtctcctggtaccagcagcccccaggcacagcccccaaactcatgatttatgaggtcagtaatcggccctcaggggtccctgatcgcttctctgggtccaagtctggcaacacggcctccctgaccatctctgggctccaggctgaggacgaggctgattattactgcagcttatatacaagcagcagcactttc IGLV3-19*01tcttctgagctgactcaggaccctgctgtgtctgtggccttgggacagacagtcaggatcacatgccaaggagacagcctcagaagctattatgcaagctggtaccagcagaagccaggacaggcccctgtacttgtcatctatggtaaaaacaaccggccctcagggatcccagaccgattctctggctccagctcaggaaacacagcttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtaactcccgggacagcagtggtaaccatct IGLV3-21*01tcctatgtgctgactcagccaccctcagtgtcagtggccccaggaaagacggccaggattacctgtgggggaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccaggcccctgtgctggtcatctattatgatagcgaccggccctcagggatccctgagcgattctctggctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggccgactattactgtcaggtgtgggacagtagtagtgatcatcc IGLV3-21*d01tcctatgtgctgactcagccaccctcagtgtcagtggccccaggaaagacggccaggattacctgtgggggaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccaggcccctgtgctggtcatctattatgatagcgaccggccctcagggatccctgagcgattctctggctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggccgactattactgtcaggtgtgggatagtagtagtgatcatcc IGLV3-22*d01tcctatgagctgacacagctaccctcggtgtcagtgtccccaggacagaaagccaggatcacctgctctggagatgtactggggaaaaattatgctgactggtaccagcagaagccaggccaggtctgatatacgagttggtgatatacgaagatagtgagcggtaccctggaatccctgaacgattctctgggtccacctcagggaacacgaccaccctgaccatcagcagggtcctgaccgaagacgaggctgactattactgtttgtctgggaatgaggacaatcc IGLV3-22*01tcctatgagctgacacagctaccctcggtgtcagtgtccccaggacagacagccaggatcacctgctctggagatgtactgggggaaaattatgctgactggtaccagcagaagccaggccaggcccctgagttggtgatatacgaagatagtgagcggtaccctggaatccctgaacgattctctgggtccacctcagggaacacgaccaccctgaccatcagcagggtcctgaccgaagacgaggctgactattactgtttgtctggggatgaggacaatcc IGLV2-23*d02cagtctgccctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaaccagcagtgatgttggtggttataactatgtctcctggtaccaacagcacccaggcaaagcccccaaactcatgatttatgatgtcagtaagcggccctcaggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgacaatctctgggctccaggctgaggacgaggctgattattactgctgctcatatgcaggtagtagcactttc IGLV2-23*02cagtctgccctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaaccagcagtgatgttgggagttataaccttgtctcctggtaccaacagcacccaggcaaagcccccaaactcatgatttatgaggtcagtaagcggccctcaggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgacaatctctgggctccaggctgaggacgaggctgattattactgctgctcatatgcaggtagtagcactttc IGLV3-25*d03tcctatgagctgacacagccaccctcggtgtcagtgtccccaggacagacggccaggatcacctgctctgcagatgcattgccaaagcaatatgcttattggtaccagcagaagccaggccaggcccctgtgctggtgatatataaagacagtgagaggccctcagggatccctgagcgattctctggctccagctcagggacaacagtcacgttgaccatcagtggagtccaggcagaagacgaggctgactattactgtcaatcagcagacagcagtggtacttatcc IGLV3-25*01tcctatgagctgatgcagccaccctcggtgtcagtgtccccaggacagacggccaggatcacctgctctggagatgcattgccaaagcaatatgcttattggtaccagcagaagccaggccaggcccctgtgctggtgatatataaagacagtgagaggccctcagggatccctgagcgattctctggctccagctcagggacaacagtcacgttgaccatcagtggagtccaggcagaagatgaggctgactattactgtcaatcagcagacagcagtggtacttatcc IGLV3-27*01tcctatgagctgacacagccatcctcagtgtcagtgtctccgggacagacagccaggatcacctgctcaggagatgtactggcaaaaaaatatgctcggtggttccagcagaagccaggccaggcccctgtgctggtgatttataaagacagtgagcggccctcagggatccctgagcgattctccggctccagctcagggaccacagtcaccttgaccatcagcggggcccaggttgaggatgaggctgactattactgttactctgcggctgacaacaatct IGLV1-36*01|Homo sapienscagtctgtgctgactcagccaccctcggtgtctgaagcccccaggcagagggtcaccatctcctgttctggaagcagctccaacatcggaaataatgctgtaaactggtaccagcagctcccaggaaaggctcccaaactcctcatctattatgatgatctgctgccctcaggggtctctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattactgtgcagcatgggatgacagcctgaatggtccIGLV5-37*01|Homo sapienscagcctgtgctgactcagccaccttcctcctccgcatctcctggagaatccgccagactcacctgcaccttgcccagtgacatcaatgttggtagctacaacatatactggtaccagcagaagccagggagccctcccaggtatctcctgtactactactcagactcagataagggccagggctctggagtccccagccgcttctctggatccaaagatgcttcagccaatacagggattttactcatctccgggctccagtctgaggatgaggctgactattactgtatgatttggccaagcaatgcttctIGLV5-39*01|Homo sapienscagcctgtgctgactcagccaacctccctctcagcatctcctggagcatcagccagattcacctgcaccttgcgcagtggcatcaatgttggtacctacaggatatactggtaccagcagaagccagggagtcttccccggtatctcctgaggtacaaatcagactcagataagcagcagggctctggagtccccagccgcttctctggatccaaagatgcttcaaccaatgcaggccttttactcatctctgggctccagtctgaagatgaggctgactattactgtgccatttggtacagcagcacttctIGLV1-40*01|Homo sapienscagtctgtgctgacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgcactgggagcagctccaacatcggggcaggttatgatgtacactggtaccagcagcttccaggaacagcccccaaactcctcatctatggtaacagcaatcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcactgggctccaggctgaggatgaggctgattattactgccagtcctatgacagcagcctgagtggttc>|IGLV7-43*01|Homo sapienscagactgtggtgactcaggagccctcactgactgtgtccccaggagggacagtcactctcacctgtgcttccagcactggagcagtcaccagtggttactatccaaactggttccagcagaaacctggacaagcacccagggcactgatttatagtacaagcaacaaacactcctggacccctgcccggttctcaggctccctccttgggggcaaagctgccctgacactgtcaggtgtgcagcctgaggacgaggctgagtattactgcctgctctactatggtggtgctcag>|IGLV1-44*01|Homo sapienscagtctgtgctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcggaagtaatactgtaaactggtaccagcagctcccaggaacggcccccaaactcctcatctatagtaataatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattactgtgcagcatgggatgacagcctgaatggtcc|IGLV5-45*03|Homo sapienscaggctgtgctgactcagccgtcttccctctctgcatctcctggagcatcagccagtctcacctgcaccttgcgcagtggcatcaatgttggtacctacaggatatactggtaccagcagaagccagggagtcctccccagtatctcctgaggtacaaatcagactcagataagcagcagggctctggagtccccagccgcttctctggatccaaagatgcttcggccaatgcagggattttactcatctctgggctccagtctgaggatgaggctgactattactgtatgatttggcacagcagcgcttct>|IGLV7-46*01|Homo sapienscaggctgtggtgactcaggagccctcactgactgtgtccccaggagggacagtcactctcacctgtggctccagcactggagctgtcaccagtggtcattatccctactggttccagcagaagcctggccaagcccccaggacactgatttatgatacaagcaacaaacactcctggacacctgcccggttctcaggctccctccttgggggcaaagctgccctgaccctttcgggtgcgcagcctgaggatgaggctgagtattactgcttgctctcctatagtggtgctcgg>|IGLV1-47*01|Homo sapienscagtctgtgctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcggaagtaattatgtatactggtaccagcagctcccaggaacggcccccaaactcctcatctataggaataatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccggtccgaggatgaggctgattattactgtgcagcatgggatgacagcctgagtggtcc >|IGLV9-49*01cagcctgtgctgactcagccaccttctgcatcagcctccctgggagcctcggtcacactcacctgcaccctgagcagcggctacagtaattataaagtggactggtaccagcagagaccagggaagggcccccggtttgtgatgcgagtgggcactggtgggattgtgggatccaagggggatggcatccctgatcgcttctcagtcttgggctcaggcctgaatcggtacctgaccatcaagaacatccaggaagaggatgagagtgactaccactgtggggcagaccatggcagtgggagcaacttcgtgtaacc >|IGLV1-51*01cagtctgtgttgacgcagccgccctcagtgtctgcggccccaggacagaaggtcaccatctcctgctctggaagcagctccaacattgggaataattatgtatcctggtaccagcagctcccaggaacagcccccaaactcctcatttatgacaataataagcgaccctcagggattcctgaccgattctctggctccaagtctggcacgtcagccaccctgggcatcaccggactccagactggggacgaggccgattattactgcggaacatgggatagcagcctgagtgctgg >|IGLV5-52*01cagcctgtgctgactcagccatcttcccattctgcatcttctggagcatcagtcagactcacctgcatgctgagcagtggcttcagtgttggggacttctggataaggtggtaccaacaaaagccagggaaccctccccggtatctcctgtactaccactcagactccaataagggccaaggctctggagttcccagccgcttctctggatccaacgatgcatcagccaatgcagggattctgcgtatctctgggctccagcctgaggatgaggctgactattactgtggtacatggcacagcaactctaagactca >|IGLV10-54*02caggcagggctgactcagccaccctcggtgtccaagggcttgagacagaccgccacactcacctgcactgggaacagcaacattgttggcaaccaaggagcagcttggctgcagcagcaccagggccaccctcccaaactcctatcctacaggaataacaaccggccctcagggatctcagagagattctctgcatccaggtcaggaaacacagcctccctgaccattactggactccagcctgaggacgaggctgactattactgctcagcattggacagcagcctcagtgctca >|IGLV6-57*01aattttatgctgactcagccccactctgtgtcggagtctccggggaagacggtaaccatctcctgcacccgcagcagtggcagcattgccagcaactatgtgcagtggtaccagcagcgcccgggcagttcccccaccactgtgatctatgaggataaccaaagaccctctggggtccctgatcggttctctggctccatcgacagctcctccaactctgcctccctcaccatctctggactgaagactgaggacgaggctgactactactgtcagtcttatgatagcagcaatca >|IGLV4-60*d03cagcctgtgctgactcaatcatcctctgcctctgcttccctgggatcctcggtcaagctcacctgcactctgagcagtgggcacagtagctacatcatcgcatggcatcagcagcagccagggaaggcccctcggtacttgatgaagcttgaaggtagtggaagctacaacaaggggagcggagttcctgatcgcttctcaggctccagctctgtggctgaccgctacctcaccatctccaacctccagtctgaggatgaggctgattattactgtgagacctgggacagtaacactca >other|IGLV4-60*03cagcctgtgctgactcaatcatcctctgcctctgcttccctgggatcctcggtcaagctcacctgcactctgagcagtgggcacagtagctacatcatcgcatggcatcagcagcagccagggaaggcccctcggtacttgatgaagcttgaaggtagtggaagctacaacaaggggagcggagttcctgatcgcttctcaggctccagctctggggctgaccgctacctcaccatctccaacctccagtctgaggatgaggctgattattactgtgagacctgggacagtaacact >|IGLV8-61*01cagactgtggtgacccaggagccatcgttctcagtgtcccctggagggacagtcacactcacttgtggcttgagctctggctcagtctctactagttactaccccagctggtaccagcagaccccaggccaggctccacgcacgctcatctacagcacaaacactcgctcttctggggtccctgatcgcttctctggctccatccttgggaacaaagctgccctcaccatcacgggggcccaggcagatgatgaatctgattattactgtgtgctgtatatgggtagtggcatttc >|IGLV4-69*01cagcttgtg ctgactcaatcgccctctgcctctgcctccctgggagcctcggtcaagctcacctgcactctgagcagtgggcacagcagctacgccatcgcatggcatcagcagcagccagagaagggccctcggtacttgatgaagcttaacagtgatggcagccacagcaagggggacgggatccctgatcgcttctcaggctccagctctggggctgagcgctacctcaccatctccagcctccagtctgaggatgaggctgactattactgtcagacctggggcactggcattca >|IGHV3-20*d01gaggtgcagctggtggagtctgggggaggtgtggtacggcctggggggtccctgagactctcctgtgcagcctctggattcacctttgatgattatggcatgagctgggtccgccaagctccagggaaggggctggagtgggtctctggtattaattggaatggtggtagcacaggttatgcagactctgtgaagggccgattcaccatctccagagacaacgccaagaactccctgtatctgcaaatgaacagtctgagagccgaggacacggccttgtattactgtgcgagaga >|IGHV1-24*d01caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttccggatacaccctcactgaattatccatgcactgggtgcgacaggctcctggaaaagggcttgagtggatgggaggttttgatcctgaagatggtgaaacaatctacgcacagaagttccagggcagagtcaccatgaccgaggacacatctacagacacagcctacatggacctgagcagcctgagatctgaggacacggccgtgtattactgtgcaacaga >|IGHV2-26*d01caggtcaccttgaaggagtctggtcctgtgctggtgaaacccacagagaccctcacgctgacctgcaccgtctctgggttctcactcagcaatgctagaatgggtgtgagctggatccatcagcccccagggaaggccctggagtggcttgcacacattttttcgaatgacgaaaaatcctacagcacatctctgaagagcaggctcaccatctccaaggacacctccaaaagccaggtggtccttaccatgaccaatatggaccctgtggacacagccacatattactgtgcacggatac >|IGKV5-2*d01gaaacgacactcacgcagtctccagcattcatgtcagcgactccaggagacaaagtcaacatctcctgcaaagccagccaagacattgatgatgatatgaactggtaccaacagaaaccaggagaagctgctattttcattattcaagaagctactactctcgttcctggaatctcacctcgattcagtggcagcgggtatggaacagattttaccctcacaattaataacatagaatctgaggatgctgcatattacttctgtctacaacatgataatttccctct >|IGKV1-9*d01gacatccagttgacccagtctccatccttcctgtctgcatctgtaggagacagagtcaccatcacttgctgggccagtcagggcattagcagttatttagcctggtatcagcaaaaaccagggaaagcccctaagctcctgatctatgctgcatccactttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcacaatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagcttaatagttaccctcc >|IGKV1D-8*d01gccatctggatgacccagtctccatccttactctctgcatctacaggagacagagtcaccatcagttgtcggatgagtcagggcattagcagttatttagcctggtatcagcaaaaaccagggaaagcccctgagctcctgatctatgctgcatccactttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagctgcctgcagtctgaagattttgcaacttattactgtcaacagtattatagtttccctcc >|IGKV3D-11*d01gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggcctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggcatcc >|IGKV1D-13*d01gccatccagttgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagggcattagcagtgctttagcctggtatcagcagaaaccagggaaagctcctaagctcctgatctatgatgcctccagtttggaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagtttaatagttaccctca >|IGKV3D-15*d01gaaatagtgatgacgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcaacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatggtgcatccatcagggccactggcatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcatcctgcagtctgaagattttgcagtttattactgtcagcagtataataactggcctcctcc >|IGKV2D-26*d01gagattgtgatgacccagactccactctccttgtctatcacccctggagagcaggcctccatgtcctgcaggtctagtcagagcctcctgcatagtgatggatacacctatttgtattggtttctgcagaaagccaggccagtctccacgctcctgatctatgaagtttccaaccggttctctggagtgccagataggttcagtggcagcgggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggattttggagtttattactgcatgcaagatgcacaagatcctcc >VκD-26*d02 V region sequence:gagattgtgatgacccagactccactctccttgtctatcacccctggagagcaggcctccatgtcctgcaggtctagtcagagcctcctgcatagtgatggatacacctatttgtattggtttctgcagaaagccaggccagtctccacgctcctgatctatgaagtttccaaccggttctctggagtgccagataggttcagtggcagcgggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggattttggagtttattactgcatgcaagatgcacaagatcctcc >|IGKV2D-28*d01gatattgtgatgactcagcctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtctagtcagagcctcctgcatagtaatggatacaactatttggattggtacctgcagaagccagggcagtctccacagctcctgatctatttgggttctaatcgggcctccggggtccctgacaggttcagtggcagtggatcaggcacagattttacactgaaaatcagcagagtggaggctgaggatgttggggtttattactgcatgcaagctctacaaactcctcc

What is claimed is:
 1. A method of obtaining an antigen-specificantibody or antigen binding fragment thereof, said antibody comprising ahuman immunoglobulin heavy (IgH) chain comprising a human IgH chainvariable region and a human IgH chain constant region, and a humanimmunoglobulin light (IgL) chain comprising a human IgL chain variableregion and a human IgL chain constant region, said antigen bindingfragment comprising said human IgH chain variable region and said humanIgL chain variable region, the method comprising: expressing saidantibody or antigen binding fragment thereof from a cell comprisingnucleic acid encoding said human IgH chain variable region and saidhuman IgL chain variable region of said antibody, wherein said antibodyof said cell comprises said human IgH chain variable region and saidhuman IgL chain variable region operably linked to a human IgH constantregion and a human IgL constant region, respectively, and wherein saidantigen binding fragment thereof comprises said human IgH chain variableregion and said human IgL chain variable region, wherein said human IgHchain variable region and said human IgL chain variable region are of atransgenic mouse contacted with said antigen, said mouse comprising inits germline a chimeric homozygous immunoglobulin heavy chain locus anda chimeric homozygous immunoglobulin light chain locus; wherein thegermline of said mouse comprises: (i) an IgH locus comprising one ormore human variable heavy (VH) gene segments, one or more human D genesegments, and one or more human joining heavy (J_(H)) gene segments atan endogenous IgH locus upstream of an enhancer and a constant (C)region comprising an endogenous IgH C gene segment, and (ii) an IgLlocus comprising four or more human variable light (VL) gene segmentsand one or more human joining light (JL) gene segments at an endogenousIgL locus upstream of an IgL constant region comprising an endogenousIgL C gene segment, wherein said IgH locus comprises in 5′ to 3′orientation said one or more human VH gene segments, said one or morehuman D gene segments, and said one or more human J_(H) gene segments,an enhancer, and said C region, wherein said intronic DNA comprises in5′ to 3′ orientation JC intronic DNA of a human IgH locus and JCintronic DNA of a mouse IgH locus, wherein said one or more J_(H) genesegments comprises a 3′ human J_(H) gene segment, wherein said 3′humanJ_(H) gene segment is less than 2 kb upstream of said mouse JC intronicDNA, wherein said human JC intronic DNA comprises human JC intronic DNAcontiguous in a human IgH locus with said 3′ human J_(H) gene segment,wherein said mouse JC intronic DNA comprises less than a complete JCintron of a mouse IgH locus, wherein said human gene segments in eachsaid Ig locus of (i) and (ii) are unrearranged and operably linked to aconstant gene segment thereof so that the mouse is capable of producingan antibody heavy chain comprising a variable domain generated byrecombination of a said one or more human J_(H) gene segments with asaid one or more D gene segments and a said one or more VH segments, andan antibody light chain comprising a variable domain generated byrecombination of a said human VL gene segment with a said one or more JLsegments, wherein said one or more human VH gene segments of the IgHlocus of (i) comprise one or more human VH gene segments selected fromthe group consisting of VH3-23*04, VH7-4-1*01, VH4-4*02, VH1-3*01,VH3-13*01, VH3-7*01, VH3-20*d01 and VH3-9*01; and wherein said four ormore human VL gene segments of the IgL chain locus of (ii) comprise fouror more human VK gene segments selected from the group consisting ofVK4-1*01, VK2-28*01, VK1D-13*d01, VK1-12*01, VK1D-12*02, VK3-20*01,VK1-17*01, VK1D-39*01, VK3-11*01, VK1 D-16*01 and VK1-9*d01.
 2. Themouse of claim 1, wherein said transgenic mouse is homozygous for saidIgH locus and said IgL locus.
 3. The method of claim 1, wherein saidmouse germline comprises human JH2*01 and/or human JH6*02, said one ormore human VH gene segments and said one or more human D gene segmentsupstream of a constant region at said endogenous heavy chain locus andhuman JK2*01 and/or human JK4*01 and said four or more human VK genesegments upstream of a constant gene segment at said endogenous lightchain locus.
 4. The method of claim 1, wherein said mouse is functionalto form rearranged human VH, D and JH gene segments and to express mRNAtranscripts encoding chimeric immunoglobulin heavy chain polypeptidecomprising a human VH region and a mouse Cμ region, wherein said mousecomprises IgH mRNA transcripts comprising IgH-VDJCμ transcriptscomprising rearranged human heavy chain V, D, and J gene segments andmouse Cμ and encoding chimeric IgH polypeptides, wherein each IgH-VDJCμtranscript encodes a human variable region comprising a CDR-H3, whereinsaid IgH-VDJCμ transcripts comprise transcripts encoding a humanvariable region comprising a CDR-H3 length of 17 amino acids andtranscripts encoding human variable region comprising a CDR-H3 length of18 amino acids, wherein the mean frequency of the group consisting ofsaid transcripts encoding CDR-H3 lengths of 17 and 18 amino acidspresent in said IgH-VDJCμ transcripts of said mouse is between 5% and10%.
 5. The method of claim 1, wherein said variable domain of saidantibody heavy chain is a product of recombination a said human VH genesegment, a human D gene segment and a human J_(H) gene segment of an IgHlocus of said (i); and/or wherein said variable domain of said antibodylight chain is a product of recombination of a said human VL genesegment and a said human JL gene segment of the IgL chain locus of (ii).6. The method of claim 1, wherein said variable domain of the heavychain is a product of recombination of a said human VH gene segment, ahuman J_(H) gene segment, and a human D gene segment, and wherein saidhuman VH gene segment is selected from the group consisting ofVH3-23*04, VH7-4-1*01, VH4-4*02, VH1-3*01, VH3-13*01, VH3-7*01,VH3-20*d01 and VH3-9*01; and/or wherein said variable domain of thelight chain is a product of recombination of a said human Vκ genesegment and a human Jκ gene segment, and wherein said human Vκ genesegment is selected from the group consisting of Vκ4-1*01, Vκ2-28*01,Vκ1D-13*d01, Vκ1-12*01, Vκ1D-12*02, Vκ3-20*01, Vκ1-17*01, Vκ1D-39*01,Vκ3-11*01, Vκ1 D-16*01 and Vκ1-9*d01.
 7. The method of claim 1, whereinsaid human VH gene segments of the IgH locus of (i) consist of allfunctional human VH gene segments; and/or wherein said human VL genesegments of the IgL chain locus of (ii) consist of all functional humanVH gene segments.
 8. A method of obtaining an antigen-specific antibodyor antigen binding fragment thereof, said antibody comprising a humanimmunoglobulin heavy (IgH) chain comprising a human IgH chain variableregion and a human IgH chain constant region, and a human immunoglobulinlight (IgL) chain comprising a human IgL chain variable region and ahuman IgL chain constant region, said antigen binding fragmentcomprising said human IgH chain variable region and said human IgL chainvariable region, the method comprising: expressing said antibody orantigen binding fragment thereof from a cell comprising nucleic acidencoding said human IgH chain variable region and said human IgL chainvariable region of said antibody, wherein said antibody of said cellcomprises said human IgH chain variable region and said human IgL chainvariable region operably linked to a human IgH constant region and ahuman IgL constant region, respectively, and wherein said antigenbinding fragment thereof comprises said human IgH chain variable regionand said human IgL chain variable region, wherein said human IgH chainvariable region and said human IgL chain variable region are of a mousecell whose genome comprises: (i) human VH, D and J_(H) gene segmentsupstream of a constant region at an endogenous heavy chain locus; and(ii) human VL and JL gene segments upstream of an constant gene segmentat an endogenous light chain locus, wherein the variable region genesegments in each said locus are operably linked to a constant genesegment of the locus so that the cell is capable of expressingimmunoglobulin heavy and light chains comprising human VH and VL domainsrespectively, wherein said heavy chain locus comprises a human 01 alleleVH gene segment capable of recombining with a human D and a human J_(H)gene segment to produce a human VH domain, wherein said light chainlocus comprises a human 01 allele VL gene segment capable of recombiningwith a human JL gene segment to produce a human VL domain, wherein themouse cell can develop into a mouse that expresses said human VH and VLdomains; wherein said heavy chain locus comprises one or more human VHgene segments selected from the group consisting of VH3-23*04,VH7-4-1*01, VH4-4*02, VH1-3*01, VH3-13*01, VH3-7*01, VH3-20*d01 andVH3-9*01, and wherein said light chain locus comprises four or morehuman VK gene segments is selected from the group consisting ofVK4-1*01, VK2-28*01, VK1 D-13*d01, VK1-12*01, VK1 D-12*02, VK3-20*01,VK1-17*01, VK1 D-39*01, VK3-1 1*01, VK1 D-16*01 and VK1-9*d01.
 9. Themethod of claim 8, wherein said mouse cell is homozygous for said IgHlocus and said IgL locus.
 10. The method of claim 8, wherein said mousecell genome comprises human JH2*01 and/or human JH6*02, said one or morehuman VH gene segments and said one or more human D gene segmentsupstream of a constant region at said endogenous heavy chain locus andhuman JK2*01 and/or human JK4*01 and said one or more human VK genesegments upstream of a constant gene segment at said endogenous lightchain locus.
 11. The method of claim 8, wherein said variable domain ofsaid antibody heavy chain is a product of recombination of a said humanVH gene segment, a human D gene segment and a human JH gene segment ofan IgH locus of said (i); and/or wherein said variable domain of saidantibody light chain is a product of recombination of a said human VLgene segment and a said human JL gene segment of the IgL chain locus of(ii).
 12. The method of claim 8, wherein said variable domain of theheavy chain is a product of recombination of a said human VH genesegment, a human JH gene segment, and a human D gene segment, andwherein said human VH gene segment is selected from the group consistingof VH3-23*04, VH7-4-1*01, VH4-4*02, VH1-3*01, VH3-13*01, VH3-7*01,VH3-20*d01 and VH3-9*01; and/or wherein said variable domain of thelight chain is a product of recombination of a said human VK genesegment and a human JK gene segment, and wherein said human VK genesegment is selected from the group consisting of VK4-1*01, VK2-28*01,VK1D-13*d01, VK1-12*01, VK1D-12*02, VK3-20*01, VK1-17*01, VK1D-39*01,VK3-11*01, VK1 D-16*01 and VK1-9*d01.
 13. The method of claim 8, whereinsaid human VH gene segments of the IgH locus of (i) consist of allfunctional human VH gene segments; and/or wherein said human VL genesegments of the IgL chain locus of (ii) consist of all functional humanVH gene segments.